Therapeutic crosslinking of cytokine receptors

ABSTRACT

The present invention relates to a compound comprising (1) a first binding moiety that binds to an Interleukin 4 receptor; and (2) a second binding moiety that binds to a cytokine receptor (e.g., an Interleukin 10 receptor, Interleukin 13 receptor, interleukin 27 receptor, interleukin 33 receptor, or transforming growth factor beta 1/2 receptor). Compounds of the disclosure cluster or crosslink the IL4 receptor and a cytokine receptor, and surprisingly, elicit unique responses in the nervous system, including unique signaling and gene expression profiles. The signaling and gene expression profiles generated IL4-cytokine fusion proteins of the disclosure are distinct from those observed in response to the combination of IL4 and the cytokine, and contribute to the superior therapeutic effects over the combination of the component parts.

CROSS REFERENCE

This application is a continuation of International Application No. PCT/EP2020/60914 which claims priority to Dutch Patent Application No. 2022982 and Dutch Patent Application No. 2022984, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING STATEMENT

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 18, 2021, is named 56780-704_301_SL.txt and is 65,718 bytes in size.

TECHNICAL FIELD

The present disclosure relates to the treatment of chronic pain, osteoarthritis, inflammatory and immune disorders.

BACKGROUND OF THE DISCLOSURE

Chronic pain is the number one reason why people seek medical advice in modern medicine. In spite of its multiple causes, chronic pain is accompanied by a cascade of biochemical reactions in the brain, spinal cord, dorsal root ganglia and peripheral nerves leading to neuroinflammation and neurodegeneration.

An example of chronic pain is neuropathic pain, which is often described as a shooting or burning pain. This type of pain can be unrelenting and severe, and often results from nerve damage or a malfunctioning nervous system. Neuropathic pain has multiple causes including amputation, chemotherapy, diabetes, HIV infection, multiple myeloma, multiple sclerosis, nerve or spinal cord compression from herniated discs or from arthritis in the spine, shingles, spine surgery, and other. Neuropathic pain also occurs with no obvious cause. Unfortunately, neuropathic pain often responds poorly to standard pain treatments and occasionally may get worse instead of better over time. For some people, it can lead to serious disability.

Another example of a chronic pain condition is osteoarthritis (OA). OA is the most common joint disorder; the majority of individuals over the age of 65 have radiographic and/or clinical evidence of OA. The most frequently affected sites are the hands, knees, hips, and spine. OA symptoms include chronic pain, significant functional impairment, stiffness, and loss of mobility. Importantly, in OA, the synovial tissue and cartilage produce pro-inflammatory cytokines, that induce chronic pain, inflammation and cartilage breakdown.

Regulatory cytokines such as Interleukin-4 (IL4), IL10 and IL13 and others, have potential in the treatment of chronic pain, inflammatory diseases, immune disorders, and other conditions.

However, cytokines as stand-alone drugs have limited therapeutic effects in chronic pain, immune, inflammatory and other biologic processes, and clinical application of these molecules for inflammatory and immune diseases has been disappointing.

It is an objective of the present disclosure to overcome one or more problems in the prior art, and the present invention discloses a new approach to improve the therapeutic application of regulatory cytokine therapy, in particular interleukin-4 (IL4), IL27, IL10, and IL13.

SUMMARY OF THE DISCLOSURE

The inventors developed a novel therapeutic approach, which relates to induction of unique signaling of cells by crosslinking of the receptors of two different cytokines. This unique signaling is neither induced by individual cytokines, nor by the combination of cytokines.

Compounds of the disclosure cluster or crosslink the IL4 receptor and a cytokine receptor, and surprisingly, elicit unique responses in the nervous system, including unique signaling and gene expression profiles. The signaling and gene expression profiles generated IL4-cytokine fusion proteins of the disclosure are distinct from those observed in response to the combination of IL4 and the cytokine, and contribute to the superior therapeutic effects over the combination of the component parts.

Bringing a receptor binding moiety corresponding to a first cytokine and a receptor binding moiety corresponding to a second cytokine together to induce unique signaling (which does not occur when both receptors are triggered simultaneously by the wild-type cytokines), can be done with various constructs, such as with a bispecific antibody or a fusion protein.

The approach according to the present disclosure, i.e., to apply cross-linking of at least two or three, preferably two receptors chosen from the group consisting of IL4R, IL10R, IL13R, IL27R, IL33R, TGFβ1R, and TGFβ2R by incorporating two or more moieties that bind to said receptors, into one molecule, for example a bispecific antibody, provides a unique treatment for chronic pain, neuro-inflammatory and neuro-degenerative diseases, and inflammatory and immune disorders. Moreover, the compounds disclosed herein, such as bispecific antibodies, have much better manufacturability, pharmacokinetic and safety profiles than cytokine-based drugs.

As an example, the first cytokine can be IL4 and the second cytokine may be IL10 or IL13, and the resulting fusion protein can be an IL4-10 fusion protein or an IL4/13 fusion protein, respectively. Administration of the resulting receptor-crosslinking agent, for example a bispecific antibody against IL4-receptor (IL4R) and IL10-receptor (IL10R) or IL13-receptor (IL13R), may completely resolve chronic pain, reduce neuro-inflammation and protect against neurodegeneration in a human or animal subject, whilst administration of a combination of the individual cytokines IL4 and IL10 or IL13 cannot.

Previously, the inventors have reported on the therapeutic effects of the IL4/IL10 fusion protein (N Eijkelkamp et al., J Neuroscience, 2016, 36 (28) 7353-7363). The inventors have now unraveled the mechanisms of action underlying the therapeutic application of the IL4-10 fusion protein according to the prior art, and found that the IL4-10 fusion protein surprisingly, contrary to the combination of IL4 and IL10, promoted clustering of the IL4 receptor (IL4R) and IL10 receptor (IL10R) on sensory neurons, which in turn promotes unique signaling pathways and gene expression profiles that can lead to a full resolution of persistent pain. The inventors now further found that an IL4-13 fusion protein induces a unique protection of neurons against the damaging effects of chemotherapeutic drugs in vitro as well as in vivo, which was dependent on cross-linking of IL4R and IL13R, as the combination of wild-type IL4 and wild-type IL13 does not provide such protection.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine for use in decreasing a sensitivity threshold of a neuronal calcium flux response of a neuron to a pro-inflammatory mediator.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine for use in decreasing a magnitude of a neuronal calcium flux response to a pro-inflammatory mediator.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine for use in decreasing a duration of a neuronal calcium flux response of a neuron to a pro-inflammatory mediator.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine for use in modulating a JAK-STAT signaling pathway in a nervous system cell.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine for use in modulating a kinomic profile of a nervous system cell in a presence of a damage associated molecular pattern (DAMP).

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine for use in modulating expression of a gene involved in calcium signaling in a nervous system cell.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine for use in decreasing ectopic neuronal activity.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to a cytokine selected from the group consisting of an interleukin 13 (IL13), an interleukin 33 (IL33), a transforming growth factor beta 1 (TGFβ1), and a transforming growth factor beta 2 (TGFβ1) for use in crosslinking an interleukin 4 receptor with a cytokine receptor.

Disclosed herein, in some aspects, is a compound that comprises an interleukin 4 polypeptide attached to an interleukin 27 for use in crosslinking an interleukin 4 receptor with an interleukin 27 receptor.

In some embodiments, the compound decreases the sensitivity threshold of the neuronal calcium flux response relative to equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, decreasing the sensitivity threshold of the neuronal calcium flux response is as determined by measuring an amount of capsaicin required to elicit a calcium flux response of a predetermined magnitude. In some embodiments, the sensitivity threshold is decreased in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the compound decreases the magnitude of the neuronal calcium flux response to the pro-inflammatory mediator relative to equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the magnitude of the neuronal calcium flux response is decreased in a presence of a damage associated molecular pattern (DAMP). 8, wherein the compound decreases the duration of the neuronal calcium flux response to the pro-inflammatory mediator relative to equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the duration of the neuronal calcium flux response is decreased in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the activity of the JAK-STAT signaling pathway is increased. In some embodiments, the activity of the JAK-STAT signaling pathway is increased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, activity of the JAK-STAT signaling pathway is modulated in a presence of a pro-inflammatory mediator. In some embodiments, activity of the JAK-STAT signaling pathway is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, a level of activity of a kinase is modulated. In some embodiments, the kinase is JAK1. In some embodiments, wherein the level of activity of the kinase is increased. In some embodiments, the level of activity of the kinase is increased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the level of activity of the kinase is modulated in a presence of a pro-inflammatory mediator. In some embodiments, the level of activity of the kinase is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, a level of phosphorylation of Annexin 2 in the nervous system cell is increased. In some embodiments, the level of phosphorylation is increased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, a level of phosphorylation of cGMP-specific 3′,5′-cyclic phosphodiesterase in the nervous system cell is decreased. In some embodiments, a level of phosphorylation is decreased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the level of phosphorylation is modulated in a presence of a pro-inflammatory mediator. In some embodiments, the level of phosphorylation is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the kinomic profile is modulated as determined by kinase array profiling relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, modulating the kinomc profile of the nervous system cell comprises increasing an activity level of tyrosine kinases relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, expression of the gene is down-regulated in the nervous system cell. In some embodiments, the expression of the gene is modulated in a presence of a pro-inflammatory mediator. In some embodiments, the expression of the gene is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the nervous system cell is a central nervous system cell. In some embodiments, the nervous system cell is a peripheral nervous system cell. In some embodiments, the nervous system cell is a neuron. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a somatosensory neuron. In some embodiments, the neuron is a visceral sensory neuron. In some embodiments, the neuron is a nociceptor. In some embodiments, the neuron is an autonomic neuron. In some embodiments, the nervous system cell is a glial cell. In some embodiments, the nervous system cell is a microglial cell. In some embodiments, the nervous system cell is an infiltrating cell. In some embodiments, the nervous system cell is an infiltrating macrophage. In some embodiments, the compound decreases the ectopic neuronal activity relative to equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, ectopic neuronal activity is decreased in a presence of a pro-inflammatory mediator. In some embodiments, ectopic neuronal activity is decreased in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the interleukin 4 polypeptide is a wild type interleukin 4. In some embodiments, the interleukin 4 polypeptide comprises an interleukin 4 derivative sequence. In some embodiments, the interleukin 4 polypeptide binds to IL-13Rα1. In some embodiments, the interleukin 4 polypeptide binds to a common gamma chain. In some embodiments, the interleukin 4 polypeptide binds to IL-4Rα. In some embodiments, the interleukin 4 polypeptide binds to IL-13Rα1, common gamma chain, and IL-4Rα. In some embodiments, the interleukin 4 polypeptide binds to IL-13Rα1 and common gamma chain with about a comparable affinity as a wild type interleukin 4. In some embodiments, the interleukin 4 polypeptide comprises a sequence that is either a wild type sequence or a derivative sequence that binds to IL-13Rα and common gamma chain with about a comparable affinity as the wild type interleukin 4. In some embodiments, the compound forms a complex with at least four receptor polypeptide chains. In some embodiments, the interleukin 4 polypeptide is a mammalian interleukin 4. In some embodiments, the interleukin 4 polypeptide is a human interleukin 4. In some embodiments, the cytokine is a mammalian cytokine. In some embodiments, the cytokine is a human cytokine. In some embodiments, the interleukin 4 polypeptide comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 11-14. In some embodiments, the interleukin 4 polypeptide comprises an amino acid sequence that is any one of SEQ ID NOs: 11-14. In some embodiments, the interleukin 4 polypeptide comprises an amino acid sequence with between 1 and 10 amino acid deletions, insertions, substitutions, or a combination thereof relative to any one of SEQ ID NOs: 11-14. In some embodiments, the cytokine comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 15-37. In some embodiments, the cytokine comprises an amino acid sequence that is any one of SEQ ID NOs: 15-37. In some embodiments, the cytokine comprises an amino acid sequence with between 1 and 10 amino acid deletions, insertions, substitutions, or a combination thereof relative to any one of SEQ ID NOs: 15-37. In some embodiments, the cytokine comprises an amino acid sequence that is a wild type cytokine sequence. In some embodiments, the cytokine a cytokine derivative sequence. In some embodiments, the interleukin 4 polypeptide and the cytokine are covalently linked. In some embodiments, the compound is a fusion protein. In some embodiments, the interleukin 4 polypeptide and the cytokine are joined by a linker. In some embodiments, the interleukin 4 polypeptide is joined to an N-terminus of the cytokine, optionally via a linker. In some embodiments, an N terminus of the interleukin 13 is joined to a C-terminus of the cytokine, optionally via a linker. In some embodiments, the compound further comprises one or more chemical modifications. In some embodiments, the chemical modification is selected from the group consisting of glycosylation, fucosylation, sialylation, and pegylation.

Disclosed herein, in some aspects, is a method of decreasing a sensitivity threshold of a neuronal response to a stimulus, comprising contacting a neuron with a compound that comprises an interleukin 4 polypeptide attached to a cytokine in an amount effective to decrease the sensitivity threshold of the neuronal response to the stimulus as determined by an amount of capsaicin required to elicit a calcium flux response of a predetermined magnitude.

Disclosed herein, in some aspects, is a method of decreasing a magnitude of a neuronal response to a stimulus, comprising contacting a neuron with a compound that comprises an interleukin 4 polypeptide attached to a cytokine in an amount effective to decrease the magnitude of the neuronal response to the stimulus as determined by measuring calcium flux in response to a predetermined concentration of capsaicin.

Disclosed herein, in some aspects, is a method of decreasing a duration of a neuronal response to a stimulus, comprising contacting a neuron with a compound that comprises an interleukin 4 polypeptide attached to a cytokine in an amount effective to decrease the duration of the neuronal response to the stimulus as determined by measuring calcium flux in response to a predetermined concentration of capsaicin.

Disclosed herein, in some aspects, is a method of decreasing ectopic neuronal activity, comprising contacting a neuron with a compound that comprises an interleukin 4 polypeptide attached to a cytokine in an amount effective to decrease ectopic activity of the neuron relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine.

Disclosed herein, in some aspects, is a method of modulating signaling in a nervous system cell, comprising contacting the nervous system cell with a compound that comprises an interleukin 4 polypeptide attached to a cytokine in an amount effective to modulate the signaling in the nervous system cell as determined by kinase array profiling of dorsal root ganglia homogenates.

Disclosed herein, in some aspects, is a method of modulating a kinomic profile of a nervous system cell, comprising contacting the nervous system cell with a compound that comprises an interleukin 4 polypeptide attached to a cytokine in an amount effective to modulate the kinomic profile in the nervous system cell as determined by kinase array profiling of dorsal root ganglia homogenates.

Disclosed herein, in some aspects, is a method of modulating gene expression in a nervous system cell, comprising contacting the nervous system cell with a compound that comprises an interleukin 4 polypeptide attached to a cytokine in an amount effective to modulate gene expression in the nervous system cell as determined by RNA sequencing.

Disclosed herein, in some aspects, is a method of crosslinking an interleukin 4 receptor with a cytokine receptor, comprising contacting a cell with a compound that comprises an interleukin 4 polypeptide attached to a cytokine selected from the group consisting of an interleukin 13, an interleukin 33, a transforming growth factor beta 1, and a transforming growth factor beta 2, thereby crosslinking the interleukin 4 receptor with the cytokine receptor, wherein the interleukin 4 polypeptide comprises a sequence that is either a wild type sequence or a variant sequence that binds to interleukin 4 receptor alpha (IL-4Rα), interleukin 13 receptor alpha (IL-13Rα), and common gamma chain with a comparable affinity as the wild type sequence.

Disclosed herein, in some aspects, is a method of crosslinking an interleukin 4 receptor with a cytokine receptor, comprising contacting a cell with a compound that comprises an interleukin 4 and an interleukin 27.

In some embodiments, the sensitivity threshold is decreased in a presence of a pro-inflammatory mediator. In some embodiments, the sensitivity threshold is decreased in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the sensitivity threshold is decreased relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the neuron is contacted with the composition for a period of time sufficient to decrease the sensitivity threshold of the neuronal response to the stimulus relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the magnitude of the neuronal response is decreased in a presence of a pro-inflammatory mediator relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the magnitude of the neuronal response is decreased in a presence of a pro-inflammatory mediator. In some embodiments, the magnitude of the neuronal response is decreased in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the neuron is contacted with the composition for a period of time sufficient to decrease the magnitude of the neuronal response to the stimulus relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the duration of the neuronal response is decreased in a presence of a pro-inflammatory mediator relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the duration of the neuronal response is decreased in a presence of a pro-inflammatory mediator. In some embodiments, the duration of the neuronal response is decreased in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the neuron is contacted with the composition for a period of time sufficient to decrease the duration of the neuronal response to the stimulus relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the stimulus comprises a pro-inflammatory mediator. In some embodiments, the stimulus comprises a pro-inflammatory cytokine, a pro-inflammatory chemokine, a vasoactive amine, a prostaglandin, a leukotriene, a thromboxane, an oxygen- and/or nitrogen-derived free radical, histamine, a Th1 cytokine, a Th2 cytokine, a Th17 cytokine, IL-1β, APRIL, IFN-α, IFN-β, IFN-γ, IL-1a, IL-1β, IL-2, IL-6, IL-8, IL-9, IL-12, IL-23, LIGHT, TNF-α, or TNF-β. In some embodiments, the stimulus comprises a damage associated molecular pattern (DAMP). In some embodiments, the stimulus comprises an anti-inflammatory mediator. In some embodiments, the stimulus comprises a drug. In some embodiments, the stimulus comprises a toxin or a toxicant. In some embodiments, the stimulus comprises a chemical stimulus. In some embodiments, the stimulus comprises a thermal stimulus. In some embodiments, the stimulus comprises a mechanical stimulus. In some embodiments, the stimulus comprises capsaicin. In some embodiments, the stimulus induces neuronal action potential firing. In some embodiments, the neuronal response comprises depolarization. In some embodiments, the neuronal response comprises action potential frequency. In some embodiments, the neuronal response comprises a transcriptional response. In some embodiments, the neuronal response comprises a signal transduction response. In some embodiments, the ectopic neuronal activity is decreased in a presence of a pro-inflammatory mediator relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the ectopic neuronal activity is decreased in a presence of a pro-inflammatory mediator. In some embodiments, the ectopic neuronal activity is decreased in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the neuron is contacted with the composition for a period of time sufficient to decrease ectopic activity of the neuron relative to a neuron contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the nervous system cell is contacted with the composition for a period of time sufficient to modulate signaling in the nervous system cell. In some embodiments, the signaling comprises a JAK-STAT signaling pathway. In some embodiments, the signaling comprises a pro-inflammatory signaling pathway. In some embodiments, the signaling comprises an innate immune system pro-inflammatory signaling pathway. In some embodiments, the signaling comprises a NOD-like receptor signaling pathway. In some embodiments, the activity of the signaling pathway is increased. In some embodiments, the activity of the signaling pathway is increased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the activity of the signaling pathway is decreased. In some embodiments, the activity of the signaling pathway is decreased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the signaling is modulated in a presence of a pro-inflammatory mediator. In some embodiments, the signaling is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, a level of activity of a kinase is modulated, wherein the kinase is JAK1. In some embodiments, a level of activity of a kinase is modulated, wherein the kinase is c-Kit. In some embodiments, a level of activity of a kinase is modulated, wherein the kinase is c-Met. In some embodiments, the level of activity of the kinase is increased. In some embodiments, the level of activity of the kinase is increased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the level of activity of the kinase is decreased. In some embodiments, the level of activity of the kinase is decreased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the level of activity of the kinase is modulated in a presence of a pro-inflammatory mediator. In some embodiments, the level of activity of the kinase is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, a level of phosphorylation of Annexin 2 in the nervous system cell is modulated. In some embodiments, a level of phosphorylation of cGMP-specific 3′,5′-cyclic phosphodiesterase in the nervous system cell is modulated. In some embodiments, the level of phosphorylation is increased. In some embodiments, the level of phosphorylation is increased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the level of phosphorylation is decreased. In some embodiments, the level of phosphorylation is decreased relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the level of phosphorylation is modulated in a presence of a pro-inflammatory mediator. In some embodiments, the level of phosphorylation is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the nervous system cell is contacted with the composition for a period of time sufficient to modulate the kinomic profile in the nervous system cell. In some embodiments, the kinomic profile is modulated as determined by kinase array profiling relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, modulating the kinomic profile of the nervous system cell comprises increasing an activity level of tyrosine kinases relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, the kinomic profile is modulated in a presence of a pro-inflammatory mediator. In some embodiments, the kinomic profile is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the nervous system cell is contacted with the composition for a period of time sufficient to modulate gene expression in the nervous system cell. In some embodiments, gene expression is modulated as determined by RNA sequencing and hierarchical clustering of the top 500 differentially-regulated genes relative to a nervous system cell that has not been contacted with the compound. In some embodiments, the gene expression is modulated as determined by RNA sequencing and hierarchical clustering of the top 500 differentially-regulated genes relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, modulating gene expression comprises modulating a global transcriptomic profile of the nervous system cell as determined by principal component analysis of RNA sequencing data relative to a nervous system cell that has not been contacted with the compound. In some embodiments, modulating gene expression comprises modulating a global transcriptomic profile of the nervous system cell as determined by principal component analysis of RNA sequencing data relative to a nervous system cell contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine. In some embodiments, expression of a gene encoding an oxidative phosphorylation enzyme is down-regulated in the nervous system cell. In some embodiments, expression of a gene involved in calcium signaling is down-regulated in the nervous system cell. In some embodiments, gene expression is modulated in a presence of a pro-inflammatory mediator. In some embodiments, gene expression is modulated in a presence of a damage associated molecular pattern (DAMP). In some embodiments, the nervous system cell is a central nervous system cell. In some embodiments, the nervous system cell is a peripheral nervous system cell. In some embodiments, the nervous system cell is a neuron. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a somatosensory neuron. In some embodiments, the neuron is a visceral sensory neuron. In some embodiments, the neuron is a nociceptor. In some embodiments, the neuron is an autonomic neuron. In some embodiments, the nervous system cell is a glial cell. In some embodiments, the nervous system cell is a microglial cell. In some embodiments, the nervous system cell is an infiltrating cell. In some embodiments, the nervous system cell is an infiltrating macrophage. In some embodiments, crosslinking is as determined by a proximity ligation assay for the interleukin receptor and the cytokine receptor. In some embodiments, the interleukin 4 polypeptide is a wild type interleukin 4. In some embodiments, the interleukin 4 polypeptide comprises an interleukin 4 derivative sequence. In some embodiments, the interleukin 4 polypeptide binds to IL-13Rα1. In some embodiments, the interleukin 4 polypeptide binds to a common gamma chain. In some embodiments, the interleukin 4 polypeptide binds to IL-4Rα. In some embodiments, the interleukin 4 polypeptide binds to IL-13Rα1, common gamma chain, and IL-4Rα. In some embodiments, the interleukin 4 polypeptide binds to IL-13Rα1 and common gamma chain with about a comparable affinity as a wild type interleukin 4. In some embodiments, the interleukin 4 polypeptide comprises a sequence that is either a wild type sequence or an IL4 derivative sequence that binds to IL-13Rα and common gamma chain with about a comparable affinity as the wild type interleukin 4. In some embodiments, the contacting, the compound form a complex with at least four receptor polypeptide chains. In some embodiments, the interleukin 4 polypeptide is a mammalian interleukin 4. In some embodiments, the interleukin 4 polypeptide is a human interleukin 4. In some embodiments, the cytokine is a mammalian cytokine. In some embodiments, the cytokine is a human cytokine. In some embodiments, the interleukin 4 polypeptide comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 11-14. In some embodiments, the interleukin 4 polypeptide comprises an amino acid sequence that is any one of SEQ ID NOs: 11-14. In some embodiments, the interleukin 4 polypeptide comprises an amino acid sequence with between 1 and 10 amino acid deletions, insertions, substitutions, or a combination thereof relative to any one of SEQ ID NOs: 11-14. In some embodiments, the cytokine comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 15-37. In some embodiments, the cytokine comprises an amino acid sequence that is any one of SEQ ID NOs: 15-37. In some embodiments, the cytokine comprises an amino acid sequence with between 1 and 10 amino acid deletions, insertions, substitutions, or a combination thereof relative to any one of SEQ ID NOs: 15-37. In some embodiments, the interleukin 4 polypeptide and the cytokine are covalently linked. In some embodiments, the compound is a fusion protein. In some embodiments, the interleukin 4 polypeptide and the cytokine are joined by a linker. In some embodiments, a C terminus of the interleukin 4 polypeptide is joined to an N-terminus of the cytokine, optionally via a linker. In some embodiments, an N terminus of the interleukin 13 is joined to a C-terminus of the cytokine, optionally via a linker. In some embodiments, the compound further comprises one or more chemical modifications. In some embodiments, the chemical modification is selected from the group consisting of glycosylation, fucosylation, sialylation, and pegylation. In some embodiments, the contacting occurs in a mammalian subject. In some embodiments, the compound is present at a concentration of at least 1 pM during the contacting. In some embodiments, the compound is present at a concentration of at least 1 nM during the contacting. In some embodiments, the compound is present at a concentration of 1 pM to 1 μM during the contacting. In some embodiments, the compound is present at a concentration of 100 pM to 100 nM during the contacting.

Disclosed herein, in some aspects is a compound that comprises an interleukin 4 polypeptide attached to a cytokine selected from the group consisting of an interleukin 13 (IL13), an interleukin 33 (IL33), a transforming growth factor beta 1 (TGFβ1), and a transforming growth factor beta 2 (TGFβ1), wherein the interleukin 4 polypeptide comprises a wild type interleukin 4 amino acid sequence.

Disclosed herein, in some aspects is a compound that comprises an interleukin 4 polypeptide attached to a cytokine selected from the group consisting of an interleukin 13 (IL13), an interleukin 33 (IL33), a transforming growth factor beta 1 (TGFβ1), and a transforming growth factor beta 2 (TGFβ1), wherein the interleukin 4 polypeptide comprises an interleukin 4 derivative that binds to interleukin 4 receptor alpha (IL-4Rα), interleukin 13 receptor alpha (IL-13Rα), and common gamma chain with a comparable affinity as a wild type interleukin 4.

Disclosed herein, in some aspects is a compound that comprises an interleukin 4 polypeptide and an interleukin 27 (IL27).

In some embodiments, the compound is a fusion protein. In some embodiments, the compound comprises the interleukin 4 polypeptide and the interleukin 13. In some embodiments, the compound comprises the interleukin 4 polypeptide and the interleukin 33. In some embodiments, the compound comprises the interleukin 4 polypeptide and the transforming growth factor beta 1. In some embodiments, the compound comprises the interleukin 4 polypeptide and the transforming growth factor beta 2. In some embodiments, the interleukin 4 polypeptide is capable of binding to IL-4Rα, IL-13Rα, and common gamma chain. In some embodiments, the compound activates signaling by a native configuration IL-4 receptor. In some embodiments, upon contacting with a cell, the compound forms a complex with at least four receptor polypeptides. In some embodiments, upon contacting a cell, the compound crosslinks an interleukin 4 receptor and a receptor for the interleukin 13, the interleukin 33, the transforming growth factor beta 1, or the transforming growth factor beta 2. In some embodiments, the compound comprises one or more chemical modifications selected from the group consisting of glycosylation, sialylation, fucosylation, and pegylation. In some embodiments, the compound is a fusion protein. In some embodiments, a nucleic acid molecule comprising a polynucleotide sequence encodes the fusion protein. In some embodiments, the polynucleotide sequence is codon optimized for expression in the cell. In some embodiments, the polynucleotide sequence is present in a nucleic acid vector. In some embodiments, the nucleic acid is present in a cell. In some embodiments, the fusion protein or nucleic acid vector is present in a pharmaceutical composition which also comprises a pharmaceutically-acceptable excipient. In some embodiments, the pharmaceutical composition is in a unit dosage form. In some embodiments, the compound is present in the pharmaceutical composition at about 50 μg to about 100 mg per mL. In some embodiments, the compound is formulated for administration as a dose of between about 0.5 μg to 1 mg per kg of body weight. In some embodiments, the compound formulated for administration as a controlled release formulation. In some embodiments, the pharmaceutical composition is formulated for administration by a parenteral, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intracerebroventricular, intracisternal, or intraparenchymal route. In some embodiments, the pharmaceutical composition is used in a method of treating a condition in need thereof, which comprises administering the compound to the subject. In some embodiments, the fusion protein is produced by culturing a cell under conditions that permit production of the fusion protein, wherein the cell comprises the polynucleotide sequence. In some embodiments, the fusion protein is harvested. In some embodiments, the fusion protein is purified from harvested culture medium.

Discloses herein, in some aspects, is a compound comprising: a first binding moiety that binds to an Interleukin 4 receptor; and a second binding moiety that binds to an Interleukin 10 receptor, wherein the first binding moiety and/or the second binding moiety is not a wild-type interleukin.

Disclosed herein, in some aspects, is a combination comprising: a first binding moiety that binds to an Interleukin 4 receptor; a second binding moiety that binds to an Interleukin 10 receptor, wherein the first binding moiety has a linker that binds the second binding moiety or wherein the second binding moiety has a linker that binds the first binding moiety, and wherein the first binding moiety and/or the second binding moiety is not a wild-type interleukin.

In some embodiments, the first binding moiety and/or the second binding moiety is a polypeptide, preferably an immunoglobulin molecule or epitope-binding fragment thereof, more preferably Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, a complementarity determining region (CDR) fragment, a single chain antibody (scFv), or single domain antibody. In some embodiments, the compound is a polypeptide, preferably a bispecific antibody, a bivalent single chain antibody, a bispecific double chain antibody, a triabody, or a tetrabody. In some embodiments, the compound or combination is able to cross-link an Interleukin 4 receptor to an Interleukin 10 receptor of a sensory neuron in vivo. In some embodiments, the first binding moiety and/or second binding moiety comprises a tag; and the linker of the first binding moiety and/or the linker of the second binding moiety is a polypeptide that binds to the tag. In some embodiments, the compound, first binding moiety and/or second binding moiety comprises a polypeptide selected from the group consisting of a signal sequence, a His-tag, and an antibody Fc fragment. In some embodiments, the compound, first binding moiety and/or second binding moiety comprises one or more chemical modifications selected from the group consisting of glycosylation, sialylation, fucosylation, and pegylation. In some embodiments, the compound or combination is comprised in a pharmaceutical composition comprising the compound or combination, and a pharmaceutically acceptable carrier. In some embodiments, the compound or combination is for use in therapeutic treatment. In some embodiments, the compound or combination is for therapeutic treatment by administration to a local compartment of a human or animal body. In some embodiments, the local compartment is an intrathecal compartment or an intraarticular compartment and/or wherein the compound or combination is administered intrathecally or intraarticularly. In some embodiments, the compound or combination is for treatment of a local condition. In some embodiments, the compound or combination is for use in the prevention or treatment of rheumatoid arthritis; osteoarthritis; other form of arthritis; pain; chronic pain; multiple sclerosis; neuro-inflammatory or neuro-degenerative disease; inflammatory bowel disease; inflammatory skin disorder; or a condition characterized by local or systemic inflammation, immune activation, and/or lymphoproliferation. In some embodiments, the compound or combination is for use in the prevention or treatment of a condition characterized by chronic pain, neuro-inflammation and/or neuro-degeneration. In some embodiments, said condition is further characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuro-inflammation, and/or neuro-degeneration. In some embodiments, said condition is selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, chemotherapy-induced allodynia, complex regional pain syndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease and Parkinson's disease, Huntington's disease, and/or amyotrophic lateral sclerosis, or multiple sclerosis. In some embodiments, the compound or composition is expressed by a cell or a cell line. In some embodiments, a nucleotide sequence encoding the compound or combination is present in a gene therapy vector that is used in the prevention or treatment of a condition characterized by chronic pain, neuro-inflammation and/or neuro-degeneration. In some embodiments, said condition is further characterized by visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuro-inflammation, and/or neuro-degeneration. In some embodiments, said condition is selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, chemotherapy-induced allodynia, complex regional pain syndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis, chronic widespread pain, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's disease and Parkinson's disease, Huntington's disease, and/or amyotrophic lateral sclerosis, or multiple sclerosis.

BRIEF DESCRIPTION OF THE FIGURES RELATED TO THE INVENTION

The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

FIG. 1. Cytokine-receptor subunits of IL10, IL4, IL13, IL33, IL27, TGFβ1, and TGFβ2 are expressed in the dorsal root ganglia of human and mouse. To evaluate whether cytokine receptors targeted by fusion proteins of the present invention are expressed by the sensory system, RNAseq data of cytokine receptor subunits for IL10, IL4, IL13, IL33, IL27, TGFβ1, and TGFβ2 in the dorsal root ganglia and spinal cord were extracted from the data base by Ray et al. (Pain 2018; 159:1325-1345) as available on www_utdallas.edu/bbs/painneurosciencelab/sensoryomics/drgtxome/?go. RNA sequencing data are expressed as transcripts per million. For comparison, data for expression of the receptors in whole blood are also given.

FIGS. 2A-2G: IL4 and IL10 receptors expressed in sensory neurons are required for analgesia induced by cross-linking IL4R and IL10R. (FIG. 2A) Expression of IL4Rα (left) and IL10Rα (right) in murine dorsal root ganglia. Persistent inflammatory pain was induced by an intraplantar injection of 20 μl of 2% carrageenan (CAR). At days 3, 4 and 5 after intraplantar injection of carrageenan, mice received intrathecal injections of mismatch (mm) or IL4R antisense oligodeoxynucleotides (asODN). At day 6 the dorsal root ganglia were obtained and analysed. (FIG. 2B) Expression of IL4R mRNA (left panel, n=13-18) and protein (right panel, n=4) in the DRG of IL4R asODN treated mice. IL4Rα mRNA levels were measured with qPCR and corrected for housekeeping genes (actin, GAPDH and HPRT). Protein expression was determined by quantifying IL4Rα immunofluorescent staining intensity. As a receptor cross-linking compound IL4-IL10 fusion protein was used. Six days after carrageenan administration mice received an intrathecal injection of 1 μg IL4-10 fusion protein (n=5) and (FIG. 2C) thermal and (FIG. 20) mechanical sensitivity was followed over time using Hargreaves and Von Frey test, respectively. Right bar graphs represent the analgesic effects of IL4-10 determined as area under the curve (AUC) between 1 and 24 hours after intrathecal injection. (FIG. 2E) Immunofluorescence staining for IL10R expression in DRGs from wild type (WT, left) or Na_(V)1.8-IL10R^(−/−) (right) mice. At six days after induction of persistent inflammatory pain WT mice and Na_(V)1.8-IL10R^(−/−) mice received an intrathecal injection of 1 μg IL4-10 fusion protein (n=13-14) and (FIG. 2F) thermal and (FIG. 2G) mechanical sensitivity was followed over time using Hargreaves or Von Frey test, respectively. Right bar graphs represent the analgesic effects of IL4-10 fusion protein determined as area under the curve (AUC) for the effect of IL4-10 fusion protein between 1 and 72 hours after intrathecal injection. Data is represented as mean±SEM. *, **, ***=p<0.05, p<0.01, and 0.001, respectively.

FIG. 3A-3C: IL4Rα and IL10Rα in sensory neurons are both required for full analgesic effects of IL4-10 fusion protein. Inflammatory pain was induced by an intraplantar injection of 20 μl of 2% carrageenan in wild type (WT) mice or Na_(V)1.8-IL10R^(−/−). At days 3, 4 and 5 after intraplantar injection mice received intrathecal injections of mismatched (mm) or IL4R antisense oligodeoxynucleotides (ODN). Six days after intraplantar injection mice received an intrathecal injection of 1 μg IL4-10 fusion protein to cross-link IL4Rα and IL10Rα (n=5 per group) and (FIG. 3A) thermal and (FIG. 3B) mechanical sensitivity was followed over time using Hargreaves or Von Frey tests, respectively. Right bar graphs represent the analgesic effects of IL4-10 fusion protein determined as area under the curve (AUC) between 1 and 24 hours after intrathecal injection. (FIG. 3C) Quantification of the total number of c-Fos positive neurons in laminae I-III of the spinal cord 24 hours after IL4-10 application. Right top panel: example c-Fos staining of the superficial dorsal horn of the spinal cord. Right bottom panels: representative pictures of c-Fos staining of the dorsal horn of naïve (i), carrageenan-injected vehicle-treated WT mice (ii), carrageenan-injected IL4-10-treated WT mice (iii) and carrageenan-injected IL4R asODN and IL4-10-treated Na_(V)1.8-IL10R^(−/−) mice (iv) (n=4-6 per group). Data is represented as mean±SEM. *, **, ***=p<0.05. 0.01, and 0.001, respectively.

FIG. 4A-4C. Crosslinking IL4R and IL13R relieves chemotherapy-induced persistent mechanical allodynia. FIG. 4A. Paclitaxel (8 mg/kg) was administered intraperitoneally to C57BL/6 mice on days 0, 2, 4 and 6 (grey symbols on the X-axis) to induce persistent chemotherapy-induced polyneuropathy. IL4-13 fusion protein (0.3 [open circle], 1 [open triangle] or 3 μg/mouse [open square]; n=4/group) or vehicle (n=4) was administered intrathecally at day 8, and the course of mechanical allodynia was followed over time using von Frey hairs. Data is represented as mean±SEM. Statistics of the data were analysed with two-way ANOVA followed by Tukey's multiple comparisons test. *, **, ***=p<0.05, p<0.01, and 0.001, 0.3 μg IL4/IL13 fusion protein versus vehicle respectively. &, && =p<0.05, p<0.01 respectively, 3 μg IL4/IL13 fusion protein versus vehicle. x=p<0.05, 1 μg IL4/IL13 fusion protein versus vehicle. FIG. 4B. On day 15, the length of intraepidermal nerve fibers in the paw skin was determined upon immunofluorescent visualization with the neuronal marker PGP9.5. The data of mice not treated with chemotherapeutic drug (black bar; n=4), or injected with paclitaxel and subsequently treated with vehicle (−; n=6) or IL4/IL3 fusion protein (IL4-13; n=4), are shown. FIG. 4C. Oxaliplatin (3 mg/kg) was daily injected intraperitoneally in mice for 5 days followed by 5 days no treatment and another 5 days of an oxaliplatin treatment cycle (grey symbols on X-axis). On the day after the last oxaliplatin injection animals received an intrathecal injection of IL4/IL13 fusion protein (0.3 μg; open circles, n=4) or the wild-type cytokines (0.15 μg; n=4, rectangles for IL4 and triangles for IL13); or vehicle only (closed circles). Pain was measured with von Frey test.

FIGS. 5A-5I: Cross-linking of IL4R and IL10R inhibits inflammatory mediator-induced sensitization of sensory neurons. Fura-2 loaded primary sensory neurons were stimulated with 30 nM capsaicin and Ca2⁺ influx was measured as the ratio of F340/F380 normalized to basal levels. Sensory neurons were stimulated overnight with (FIG. 5A) TNFα (50 ng/ml, n=157-244) or (FIG. 5D) PGE2 (1 μM, n=119-188) in the absence or presence of the IL4-10 fusion protein (100 ng/ml, 3 nM). Total calcium fluxes were quantified by determining the maximal amplitude (FIG. 5B, FIG. 5E) of capsaicin-evoked Ca2+ responses (applied at time point 1 minute) and the area under curve (AUC; FIG. 5C, FIG. 5F) of capsaicin-evoked Ca2⁺ influx over 5 minutes. (FIG. 5G) Sensory neurons were stimulated overnight with TNFα (50 ng/ml) and IL4-10 fusion protein (100 ng/ml) in the presence of receptor-blocking antibodies (2 μg/ml) against the IL4 receptor (αIL4R) and the IL10 receptor (αIL10R). Inhibition of TNFα-sensitization was measured as percentage of AUC of capsaicin-evoked Ca²⁺ response over 5 minutes. Asterisks represent significant differences compared to the TNFα-sensitized neuronal response. (n=51-164) (FIG. 5H, FIG. 5I) Sensory neurons were stimulated overnight with TNFα (50 ng/ml) in combination with different concentrations (0.6, 6 and 60 nM) of IL4-10 fusion protein or equimolar doses of the combination of both recombinant cytokines. Inhibition of TNFα-sensitization was measured as the percentage of the (FIG. 5H) amplitude or (FIG. 5I) AUC of capsaicin-evoked Ca2⁺ response over 5 minutes (n=107-244). Data is represented as mean±SEM. *, **, ***=p<0.05, 0.01 and 0.001, respectively (n=51-244 from at least 3 different cultures).

FIG. 6. Cross-linking of IL4R and IL13R is required to protect cultured neurons against oxaliplatin-induced damage. Primary sensory neurons were cultured and treated overnight with oxaliplatin (5 μg/ml). Neuronal damage was then quantified by measuring the neurite length upon β3-tubulin staining. Vehicle (−) or IL4/IL13 fusion protein or the combination of IL4 and IL13 (IL4+IL13) were added at equimolar concentrations during incubation with the chemotherapeutic drug. Neurons cultured in absence of oxaliplatin and cytokines are shown for comparison (black bar).

FIGS. 7A-7B: IL4-10 fusion protein induces heterologous receptor clustering in sensory neurons. (FIG. 7A) Cultured sensory neurons were treated for 15 minutes with IL4-10 fusion protein (100 ng/ml; right column), the combination of IL4 and IL10 (50 ng/ml each; middle column) or vehicle (left column). After fixation, a proximity ligation assay (PLA) for IL4R and IL10R was performed (red) and combined with immunofluorescent staining for anti-βIII-tubulin to identify sensory neurons (green). Presence of red fluorescence indicates that IL4R and IL10R are at less than 51 nm in proximity to each other. (FIG. 7B) Intensity of PLA staining was quantified using Image J software. For each condition 3 to 6 pictures of each of 2 different experiments were analysed. Data is represented as mean±SD. *=p<0.05.

FIGS. 8A-8F. Kinome activity profile in the DRG of mice with chronic pain after cross-linking IL4R and IL10R with a fusion protein. Mice received an intraplantar injection of 20 μl of 2% carrageenan and 6 days later received an intrathecal injection with either vehicle, the combination of IL4+IL10 (0.5 μg each), or IL4R-IL10R cross-linking compound, i.e., IL4-10 fusion protein (1 μg). One hour after intrathecal injection DRGs were isolated and DRG homogenates were subjected to PAMGENE analysis. (FIGS. 8A-8D) List of peptides from the PAM chips which are differentially regulated based on one-way ANOVA analysis. Blue colour indicates diminished phosphorylation of peptide substrates, and red colour indicates increased phosphorylation of peptides in DRG lysates of IL4-IL10-treated mice compared to those treated with IL4+IL10. Black color indicates no significant changes. (A/B) Peptides that are significantly different in the PTK chip. (FIG. 8A) List of PTK target peptides differentially regulated between IL4-10 and IL4+IL10-treated animals compared to control-treated animals. (FIG. 8B) List of PTK target peptides differentially regulated between IL4-10-treated animals compared to IL4+IL10 treated animals. (C/D) Peptides that are significantly differently phosphorylated in the STK chip compared to vehicle. (FIG. 8C) List of STK target peptides differentially regulated between IL4-10 and IL4+IL10 treated animals compared to control-treated animals. (FIG. 8D) List of STK target peptides differentially regulated between IL4-10-treated animals and IL4+IL10 treated animals. (FIG. 8E) Predicted upstream kinases that can be inferred from the peptide substrates differentially phosphorylated on the PAM chips. Unpaired t-test comparison between samples from IL4-10-treated animals compared to IL4+IL10-treated animals (n=5 animals per group). (Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www_cellsignal.com)). (FIG. 8F) Enriched GeneGo process pathway analysis based on peptides that were significantly differentially phosphorylated after t-test comparison between IL4-10 and IL4+IL10-treated animals. The height of the histogram corresponds to the p-values of signalling pathways that are significantly enriched by differentially phosphorylated peptides. (FIG. 8G) Top 10 predicted Protein Tyrosine kinases differentially regulated between samples from IL4-10-treated animals compared to IL4+IL10-treated animals. The graph is sorted with the highest specificity scores at the top, and the lowest specificity scores at the bottom (FIG. 8H) top 10 predicted Serine/threonine kinases differentially regulated between samples from IL4-10-treated animals compared to IL4+IL10-treated animals. The graph is sorted with the highest specificity scores at the top, and the lowest specificity scores at the bottom

FIGS. 9A-9F. Transcriptome analysis of the DRG after intrathecal injection of IL4-10. Persistent inflammatory pain was induced by an intraplantar injection of 20 μl of 2% carrageenan. Six days later mice received an intrathecal injection of vehicle (control), IL4+IL10 (0.5 μg each) or IL4-10 fusion protein (1 μg). Six hours after intrathecal injection, lumbar DRGs (L3-L5) were isolated and subjected to RNA-sequencing. (FIG. 9A) Principal component analysis (PCA) of the differentially expressed genes in all pair-wise comparisons of different animal. (FIG. 9B) Hierarchical clustering heat map of the expression levels of the top 500 differentially expressed genes (based on adjusted p-values). (FIG. 9C) Venn diagram showing the number of differentially expressed genes in IL4-10 fusion protein or IL4+IL10 treated animals compared to vehicle-treated animals. (FIG. 9D) The volcano plot shows the adjusted p-values and fold changes for all transcripts in IL4+IL10-treated mice compared to IL4-10-treated mice. Differential expression of genes (FDR corrected p-value<0.05) was determined using DESeq2 package in R. (FIG. 9E) Top 25 pathway analysis of genes differentially regulated between IL4-10 fusion protein and the combination of IL4+IL10 (toppgene.cchmc.org/). (FIG. 9F) Pathway enrichment analysis of the differentially regulated transcript (IL4-10 versus IL4+II10) to identifying potential signaling pathways upstream of the differentially regulated genes

FIGS. 10A-10E. Interrogation of role of the role of kinases in the superior analgesic effect of IL4-10. (FIG. 10A) Sensory neurons were treated in vitro for 10, 30 or 60 minutes with IL4-10 fusion protein (100 ng/ml; light grey columns), the combination of IL4 and IL10 (50 ng/ml each; dark grey columns) or vehicle (black column). Cells were stained for pJAK1 and fluorescence intensity of individual cells were measured. Data are obtained in 4 different independent primary neuronal cultures. (B-E) Persistent inflammatory pain was induced by an intraplantar injection of 20 μl of 2% carrageenan. (B, C) At days 5, 6 and 7 after intraplantar injection mice received Ruxolitinib (JAK1/2 inhibitor; n=8) or vehicle orally (n=4). Six days after intraplantar injection mice received an intrathecal injection of 1 μg IL4-10 fusion protein (n=8) or PBS as vehicle (n=4) and (FIG. 10B) thermal and (FIG. 10C) mechanical sensitivity was followed overtime using Hargreaves or Von Frey tests, respectively. Right bar graphs represent the total analgesic effects of IL4-10 determined as area under the curve (AUC) between 1 and 72 hours after intrathecal injection. *p<0.05 IL4-10: Vehicle versus IL4-10: Ruxolitinib-treated mice. #p<0.05 IL4-10: Vehicle versus IL4+IL10: Vehicle-treated mice. (D, E) Six days after intraplantar injection of carrageenan mice received an intrathecal injection of 1 μg IL4-10 fusion protein and (FIG. 10D) thermal and (FIG. 10E) mechanical sensitivity was followed over time using Hargreaves or Von Frey tests, respectively. To inhibit c-kit or both c-kit and PDGFR mice received respectively intraperitoneal injections of Dasatinib (n=4), or Masitinib (n=4) at days 5, 6 and 7 after intraplantar carrageenan injection. To inhibit MET mice were orally administered with the MET inhibitor JNJ-38877605 (n=5) or vehicle. Bar graphs represent the analgesic effects of IL4-10 determined as area under the curve (AUC) between 1 and 72 hours after intrathecal injection. Data is represented as mean±SEM. *, **, ***=p<0.05. 0.01, and 0.001, respectively.

FIG. 11. An IL4-containing fusion protein of the disclosure elicits a distinct kinase activity profile in dorsal root ganglia (DRG) cells compared to a combination of unlinked cytokines. PamGene kinase activity profiling was performed to assess global protein tyrosine kinases (PTK) activity in homogenates of lumbar DRGs isolated from mice with persistent paclitaxel-induced peripheral neuropathy after IL4/IL13 fusion protein, IL4+IL13 (combination of unlinked cytokines), and vehicle administration. Kinomic profiles were assessed at 60 minutes after intrathecal administration of the IL4/IL13 fusion protein, the combination of cytokines, or vehicle (PBS). Peptides are shown that were differentially phosphorylated based on one-way ANOVA analysis between IL4/IL13, IL4+IL13, and vehicle-treated mice compared to naive mice (untreated; no paclitaxel, no intrathecal injection). Black indicates no significant changes, while color indicates decreased phosphorylation.

FIG. 12. Altered kinase activity in dorsal root ganglia (DRG) cells of female mice treated with IL4/IL13 compared to a combination of unlinked cytokines. PamGene kinase activity profiling was performed to assess global protein tyrosine kinases (PTK) activity in homogenates of lumbar DRGs isolated from female mice with persistent paclitaxel-induced neuropathy after IL4/IL13 fusion protein or IL4+IL13 (combination of unlinked cytokines) administration. The graph shows the predicted upstream kinases inferred from the differentially phosphorylated peptide substrates identified by unpaired t-test comparison between samples from IL4/IL13 fusion protein-treated females and IL4+IL13-treated females (n=3 animals per group). The graph is sorted with the highest specificity scores at the top, and the lowest specificity scores at the bottom.

FIG. 13. Altered kinase activity in dorsal root ganglia (DRG) cells of male mice treated with IL4/IL13 compared to a combination of unlinked cytokines. PamGene kinase activity profiling was performed to assess global protein tyrosine kinases (PTK) activity in homogenates of lumbar DRGs isolated from male mice with persistent paclitaxel-induced neuropathy after IL4/IL13 fusion protein or IL4+IL13 (combination of unlinked cytokines) administration. The graph shows the predicted upstream kinases inferred from the differentially phosphorylated peptide substrates identified by unpaired t test comparison between samples from IL4/IL13 fusion protein-treated males and IL4+IL13-treated males (n=3 animals per group). The graph is sorted with the highest specificity scores at the top, and the lowest specificity scores at the bottom.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides compounds (e.g., polypeptide constructs, fusion proteins) that comprise an interleukin 4 (IL4) directly or indirectly linked to an additional cytokine, for example, a cytokine selected from the group consisting of an interleukin 10 (IL10). an interleukin 13 (IL13), an interleukin 27 (IL27), an interleukin 33 (IL33), a transforming growth factor beta 1 (TGFβ1), a transforming growth factor beta 2 (TGFβ2), and an additional IL4.

Compounds of the disclosure can cluster or crosslink the IL4 receptor and a cytokine receptor, and surprisingly, elicit unique responses in the nervous system, including unique signaling and gene expression profiles. The signaling and gene expression profiles generated IL4-cytokine fusion proteins of the disclosure are distinct from those observed in response to the combination of IL4 and the cytokine, and can contribute to superior therapeutic effects over the combination of the component parts.

In some embodiments, compounds (e.g., polypeptide constructs, fusion proteins) disclosed herein elicit unique and favorable responses in nervous system cells compared to equivalent amounts of IL4 and the cytokine individually. The disclosed compounds (e.g., polypeptide constructs, fusion proteins) can be useful for treating a condition disclosed herein in a subject in need thereof, including, for example, achieving superior therapeutic effects compared to equivalent amounts of IL4 and the cytokine administered separately or in combination (e.g., as a combination of unlinked cytokines).

When describing a cytokine, the term “wild type” refers to a cytokine with an amino acid sequence that is naturally occurring and encoded by a germline genome of a given species. A species can have one wild type sequence, or two or more wild type sequences (for example, with one canonical wild type sequence and one or more non-canonical wild type sequences). A wild type cytokine sequence can include a sequence that is truncated at the N and/or C terminus relative to the sequence encoded by an open reading frame. A wild type cytokine sequence can be a mature form of a cytokine that has been processed to remove N-terminal and/or C-terminal residues. A wild type cytokine can lack a signal peptide or can include a signal peptide (e.g., a signal peptide can be added to the N-terminus of the wild type cytokine).

When describing a cytokine, the term “derivative” refers to a cytokine with an amino acid sequence that differs from a wild type sequence by one or more amino acids, for example, containing one or more amino acid insertions, deletions, or substitutions relative to a wild type sequence. A cytokine derivative binds to at least one subunit of the corresponding native receptor for the wild type cytokine and elicits signaling and/or cytokine activity. The binding affinity, signaling, and/or cytokine activity of a cytokine derivative can be the same or different than the corresponding wild type cytokine.

The term “nervous system cell” refers to a cell that is found within the central nervous system or peripheral nervous system. A nervous system cell can be a neuron, a central nervous system cell, a peripheral nervous system cell, a glial cell, a microglial cell, an astrocyte, a Schwann cell, a satellite glial cell, an oligodendrocyte, an infiltrating cell, an infiltrating immune cell, an infiltrating myeloid cell, an infiltrating lymphoid cell, an infiltrating macrophage, an infiltrating neutrophil, an infiltrating lymphocyte, an infiltrating T cell, an infiltrating B cell, or an infiltrating natural killer cell. A neuron can be, for example, a sensory neuron, a somatosensory neuron, a visceral sensory neuron, a nociceptor, and/or an autonomic neuron.

“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty=50 (nucleotides)/8 (proteins) and gap extension penalty=3 (nucleotides)/2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif. 92121-3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively, percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc. Sequence identity can refer to the sequence identity over the entire length of the sequence.

Interleukin 4:

A compound (e.g., polypeptide construct, fusion protein) disclosed herein can comprise an IL4 protein, or a variant, derivative or fragment thereof operably connected or directly or indirectly fused to at least one additional cytokine or a variant, derivative or fragment thereof. The IL4 protein can be a mammalian IL4 protein, such as a human IL4, or mouse IL4. Non-limiting examples of amino acid sequences of IL4 are set forth in SEQ ID NOs: 11-14.

TABLE 1 non-limiting examples of human IL4 sequences of the disclosure SEQ ID NO: SEQUENCE 11 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYS HHEKDTRCLGATAQQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLE NFLERLKTIMREKYSKCSS 12 HKCDITLQEIIKTLNSLTEQKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQ FHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKC SS 13 HKRDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYS HHEKDTRCLGATAQQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLE NFLERLKTIMREKYSKCSS 14 HKRDITLQEIIKTLNSLTEQKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQ FHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKC SS

Variants of IL4 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to any one of SEQ ID NOs: 11-14, for example, over the entire length. Amino acid sequence identity can be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters, e.g., as disclosed herein. Variants and derivatives also include proteins having IL4 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of any one of SEQ ID NOs: 11-14. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL4 of the disclosure (e.g., an IL4 variant, derivative, or fragment thereof) can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions relative to an IL4 sequence disclosed herein (e.g., a wild type IL4 sequence).

In some embodiments, an IL4 of the disclosure (e.g., an IL4 variant, derivative, or fragment thereof) can comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL4 sequence disclosed herein (e.g., a wild type IL4 sequence).

In some embodiments, an IL4 sequence of the disclosure (e.g., an IL4 variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions relative to an IL4 sequence disclosed herein (e.g., a wild type IL4 sequence).

In some embodiments, an IL4 sequence of the disclosure (e.g., an IL4 variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to an IL4 sequence disclosed herein (e.g., a wild type IL4 sequence). An amino acid substitution can be a conservative or a non-conservative substitution. The one or more amino acid substitutions, deletions, or insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions can be contiguous, non-contiguous, or a combination thereof.

An IL4 of the disclosure can comprise a wild type IL4 sequence. Non-limiting examples of wild type IL4 sequences include SEQ ID NOs: 11-14. SEQ ID NO: 11 can be a canonical wild type IL4 sequence of the disclosure.

An IL4 of the disclosure can comprise an IL4 variant, derivative, or fragment thereof with one or more amino acid substitutions. For example, an IL4 variant, derivative, or fragment thereof can comprise an amino acid substitution at position K117, T118, R121, E122, Y124, S125, S128, S129, or a combination thereof of SEQ ID NO: 11. In some embodiments, an IL4 variant, derivative, or fragment thereof comprises a substitution that is K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S125R, S129A, or a combination thereof relative to SEQ ID NO: 11. In some embodiments, an IL4 variant, derivative, or fragment thereof comprises the substitutions K117R, T118V, R121Q, E122S, Y124W, S125F, S128G, and S129A relative to SEQ ID NO: 11. In some embodiments, an IL4 variant, derivative, or fragment thereof comprises the substitutions R121D and Y124D relative to SEQ ID NO: 11.

In some embodiments, an IL4 variant, derivative, or fragment thereof does not contain a substitution at position K117, T118, R121, E122, Y124, S125, S128, or S129, relative to SEQ ID NO: 11. In some embodiments, an IL4 variant, derivative, or fragment thereof does not contain a K117R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S125R, or S129A substitution.

In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure binds to an IL4 receptor subunit with about a comparable affinity as a wild type IL4 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold increased affinity compared to a wild type IL4 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold decreased affinity compared to a wild type IL4 sequence.

For example, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 13 receptor alpha 1 (IL-13Rα1), common gamma chain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereof, e.g. with about a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 with about a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to common gamma chain with about a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-4Rα with about a comparable affinity as a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and common gamma chain with about a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα with about a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to common gamma chain and IL-4Rα with about a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1, common gamma chain, and IL-4Rα with about a comparable affinity as a wild type IL4.

In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure binds to an IL4 receptor subunit with at least a comparable affinity as a wild type IL4 sequence. For example, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 13 receptor alpha 1 (IL-13Rα1), common gamma chain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with at least a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 with at least a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to common gamma chain with at least a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-4Rα with at least a comparable affinity as a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and common gamma chain with at least a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα with at least a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to common gamma chain and IL-4Rα with at least a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1, common gamma chain, and IL-4Rα with at least a comparable affinity as a wild type IL4.

In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure binds to an IL4 receptor subunit with at most a comparable affinity as a wild type IL4 sequence. For example, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 13 receptor alpha 1 (IL-13Rα1), common gamma chain, interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with at most a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 with at most a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to common gamma chain with at most a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-4Rα with at most a comparable affinity as a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and common gamma chain with at most a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα with at most a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to common gamma chain and IL-4Rα with at most a comparable affinity as a wild type IL4. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1, common gamma chain, and IL-4Rα with at most a comparable affinity as a wild type IL4.

In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof can bind to an IL-13Rα1 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof can bind to an IL-13Rα1 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL4 sequence.

In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof can bind to a common gamma chain with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof can bind to a common gamma chain with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL4 sequence.

In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof can bind to an IL-4Rα with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL4 sequence. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof can bind to an IL-4Rα with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL4 sequence.

In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can activate a native IL4 receptor. A native IL4 receptor can be, for example, a receptor comprising an IL-13Rα1 subunit and an IL-4Rα subunit, or a common gamma chain subunit and an IL-4Rα subunit. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can activate a native IL4 receptor when present in a fusion protein. In some embodiments, an IL4 or IL4 variant, derivative, or fragment thereof of the disclosure can activate a native IL4 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native IL4 receptor when present in a fusion protein.

In some embodiments, a polypeptide of the disclosure does not contain IL4. In some embodiments, a polypeptide of the disclosure does not contain SEQ ID NO: 11.

Interleukin 10:

A compound (e.g., polypeptide construct, fusion protein) may comprise a monomeric or multimeric (for instance homodimeric) IL10 protein, or a variant, derivative, or fragment thereof operably connected or directly or indirectly fused to an IL4 protein, or a variant, derivative or fragment thereof. The IL10 protein can be a mammalian IL10 protein, such as a human IL10, or mouse IL10. One amino acid sequence representing IL10 is set forth in SEQ ID NO: 15.

TABLE 2 non-limiting examples of human IL10 sequences of the disclosure SEQ ID NO: SEQUENCE 15 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLL EDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRR CHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

Variants of IL10 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO: 15, for example, over the entire length. Amino acid sequence identity can be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as disclosed herein. Variants, derivatives, and fragments thereof also include proteins having IL10 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO: 15. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL10 of the disclosure (e.g., an IL10 variant, derivative, or fragment thereof) can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions relative to an IL10 sequence disclosed herein (e.g., a wild type IL10 sequence).

In some embodiments, an IL10 of the disclosure (e.g., an IL10 variant, derivative, or fragment thereof) can comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL10 sequence disclosed herein (e.g., a wild type IL10 sequence).

In some embodiments, an IL10 sequence of the disclosure (e.g., an IL10 variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions relative to an IL10 sequence disclosed herein (e.g., a wild type IL10 sequence).

In some embodiments, an IL10 sequence of the disclosure (e.g., an IL10 variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to an IL10 sequence disclosed herein (e.g., a wild type IL10 sequence). An amino acid substitution can be a conservative or a non-conservative substitution. The one or more amino acid substitutions, deletions, or insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions can be contiguous, non-contiguous, or a combination thereof.

An IL10 of the disclosure can comprise a wild type IL10 sequence. A non-limiting examples of a wild type IL10 sequences is SEQ ID NO: 15. SEQ ID NO: 15 can be a canonical wild type IL10 sequence of the disclosure.

An IL10 of the disclosure can comprise an IL10 variant, derivative, or fragment thereof with one or more amino acid substitutions. For example, an IL10 variant, derivative, or fragment thereof can comprise an amino acid substitution at position I87, A89, H109, R110, F111, Y153, M156, or a combination thereof of SEQ ID NO: 15. In some embodiments, an IL10 variant, derivative, or fragment thereof comprises a substitution that is M156, F111S, I87A, I87G, A89D, H109D, R110D, Y153D, M156D, A89D, H109E, R110E, Y153E, M156E, or a combination thereof relative to SEQ ID NO: 15.

In some embodiments, an IL10 variant, derivative, or fragment thereof does not contain a substitution at position I87, A89, H109, R110, F111, Y153, or M156 relative to SEQ ID NO: 15. In some embodiments, an IL10 variant, derivative, or fragment thereof does not contain a M156, F111S, I87A, I87G, A89D, H109D, R110D, Y153D, M156D, A89D, H109E, R110E, Y153E, or M156E substitution.

In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure binds to an IL10 receptor subunit with about a comparable affinity as a wild type IL10 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold increased affinity compared to a wild type IL10 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold decreased affinity compared to a wild type IL10 sequence.

For example, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 10 receptor 1 (IL-10R1), interleukin 10 receptor 2 (IL-10R2), or a combination thereof with about a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to IL-10R1 with about a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to IL-10R2 with about a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to IL-10R1 and IL-10R2 with about a comparable affinity as a wild type IL10.

In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL10 receptor subunit with at least a comparable affinity as a wild type IL10. For example, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to IL-10R1, IL-10R2, or a combination thereof with at least a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL-10R1 with at least a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL-10R2 with at least a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL-10R1 and an IL-10R2 with at least a comparable affinity as a wild type IL10.

In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL10 receptor subunit with at most a comparable affinity as a wild type IL10. For example, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to IL-10R1, IL-10R2, or a combination thereof with at most a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL-10R1 with at most a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL-10R2 with at most a comparable affinity as a wild type IL10. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can bind to an IL-10R1 and an IL-10R2 with at most a comparable affinity as a wild type IL10.

In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof can bind to an IL-10R1 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL10 sequence. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof can bind to an IL-10R1 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL10 sequence.

In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof can bind to an IL-10R2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL10 sequence. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof can bind to an IL-10R2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL10 sequence.

In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can activate a native IL10 receptor. A native IL10 receptor can be, for example, a receptor comprising an IL-10R1 subunit and an IL-10R2 subunit. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can activate a native IL10 receptor when present in a fusion protein. In some embodiments, an IL10 or IL10 variant, derivative, or fragment thereof of the disclosure can activate a native IL10 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native IL10 receptor when present in a fusion protein.

In some embodiments, a compound (e.g., fusion protein or polypeptide construct) of the disclosure does not contain IL10. In some embodiments, a polypeptide of the disclosure does not contain SEQ ID NO: 15.

Interleukin 13:

A compound (e.g., polypeptide construct, fusion protein) disclosed herein can comprise an IL13 protein, or a variant, derivative, or fragment thereof operably connected or directly or indirectly fused to an IL4 protein, or a variant, derivative or fragment thereof. The IL13 protein can be a mammalian IL13 protein, such as a human IL13, or mouse IL13. Non-limiting examples of amino acid sequences representing IL13 are set forth in SEQ ID NOs 16-23.

TABLE 3 non-limiting examples of human IL13 sequences of the disclosure SEQ ID NO: SEQUENCE 16 PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVS GCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREG QFN 17 GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSG CSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQ FN 18 SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINV SGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFRE GQFN 19 LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCA ALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFREGQFN 20 PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVS GCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREG RFN 21 GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSG CSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGR FN 22 SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINV SGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFRE GRFN 23 LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCA ALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFREGRFN

Variants of IL13 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity, to any one of SEQ ID NOs: 16-23, for example, over the entire length. Amino acid sequence identity can be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as disclosed herein. Variants, derivatives, and fragments thereof also include proteins having IL13 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of any one of SEQ ID NOs: 16-23. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL13 (e.g., an IL13 variant, derivative, or fragment thereof) of the disclosure can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions relative to an IL13 sequence disclosed herein (e.g., a wild type IL13 sequence).

In some embodiments, an IL13 (e.g., an IL13 variant, derivative, or fragment thereof) of the disclosure can comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL13 sequence disclosed herein (e.g., a wild type IL13 sequence).

In some embodiments, an IL13 sequence (e.g., an IL13 variant, derivative, or fragment thereof) of the disclosure can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions relative to an IL13 sequence disclosed herein (e.g., a wild type IL13 sequence).

In some embodiments, an IL13 sequence (e.g., an IL13 variant, derivative, or fragment thereof) of the disclosure can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to an IL13 sequence disclosed herein (e.g., a wild type IL13 sequence). An amino acid substitution can be a conservative or a non-conservative substitution. The one or more amino acid substitutions, deletions, or insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions can be contiguous, non-contiguous, or a combination thereof.

An IL13 of the disclosure can comprise a wild type IL13 sequence. Non-limiting examples of wild type IL13 sequences include any one of SEQ ID NOs: 16-23. SEQ ID NO: 20 can be a canonical wild type IL13 sequence of the disclosure.

An IL13 of the disclosure can comprise an IL13 variant, derivative, or fragment thereof with one or more amino acid substitutions. For example, an IL13 variant, derivative, or fragment thereof can comprise an amino acid substitution at position L10, E12, R11, I14, E15, E16, V18, R65, S68, R86, D87, T88, K89, D98, L101, L103, K104, K105 L106, F107, R108, R111, F114, N113, or a combination thereof of SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises a substitution that is L10F; L10I; L10V; L10A; L10D; L10T; L10H; R11S; R11N; R11H; R11L; R11I; I14L; I14F; I14V; I14M; V18L; V18F; V18I; E12A; R65D; R86K; R86T; R86M; D87E; D87K; D87R; D87G; D87S; T88S, T881; T88K; T88R; K89R; K89T; K89M; L101F; L101I; L101Y; L101H; L101N; K104R; K104T; K104M; K105T; K105A; K105R; K105E; F107L; F107I; F107V; F107M; R108K; R108T; R108M; E12K, E12I, E12C, E12S, E12R, E12Y, E12D, E15K, E16K, R65D, S68D, D98K, L101A, L103A, K104D, K105D, L106A, F107Y, R108D, R111D, F114D, N113D, or a combination thereof relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10H, R86T, D87G, T88R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10V, K89R, L101 N, K105E, and R108T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10D, R11I, V18I, R86K, D87K, K89R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10V, K89R, L101 N, K105E, and R108T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions R11S, I14M, T88S, L101 N, K105A, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10H, R86T, D87G, T88R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10T or L10D; R11I; V18I; R86K; D87K or D87G; T88S; K89R; L101Y; K104R; K105T; and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10A or L10V; R86T; D87G; T88K; K89R; L101N; K104R; K105A or K105E; and R108K or R108T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10V, V18I, D87S, D88S, L101F, K104R, and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10V, V18I, D87S, T88S, L101F, K104R, and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10V or L10I; D87S; T88S; K89R; L101H or L101F; K104R; and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10I; V18I; R86T; D87G; T88S; K89R; L101Y, L101H; K104R; and K105A relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions L10V; V18I; D87S; T88S; L101F; K104R; and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions V18I, R86T, D87G, T88S, L101Y, K104R, and K105A relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, and F107M relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12K and S68D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12K and R108D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12K and R111D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12Y and R65D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12Y and S68D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12K, R65D and S68D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12Y, R65D and S68D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof comprises the substitutions E12K, R65D, S68D and R111D relative to SEQ ID NO: 16 or SEQ ID NO: 20.

In some embodiments, an IL13 variant, derivative, or fragment thereof does not contain a substitution at position L10, E12, R11, I14, E15, E16, V18, R65, S68, R86, D87, T88, K89, D98, L101, L103, K104, K105 L106, F107, R108, R111, F114, or N113 relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or fragment thereof does not contain a L10F; L10I; L10V; L10A; L10D; L10T; L10H; R11S; R11N; R11H; R11L; R11I; I14L; I14F; I14V; I14M; V18L; V18F; V18I; E12A; R65D; R86K; R86T; R86M; D87E; D87K; D87R; D87G; D87S; T88S, T881; T88K; T88R; K89R; K89T; K89M; L101F; L101I; L101Y; L101H; L101N; K104R; K104T; K104M; K105T; K105A; K105R; K105E; F107L; F107I; F107V; F107M; R108K; R108T; R108M; E12K, E12I, E12C, E12S, E12R, E12Y, E12D, E15K, E16K, R65D, S68D, D98K, L101A, L103A, K104D, K105D, L106A, F107Y, R108D, R111D, F114D, or N113D substitution.

In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure binds to an IL13 receptor subunit with about a comparable affinity as a wild type IL13 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold increased affinity compared to a wild type IL13 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold decreased affinity compared to a wild type IL13 sequence.

For example, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 13 receptor alpha 1 (IL-13Rα1), interleukin 13 receptor alpha 2 (IL-13Rα2), interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with about a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 with about a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα2 with about a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-4Rα with about a comparable affinity as a wild type IL13 sequence. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-13Rα2 with about a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα with about a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα2 and IL-4Rα with about a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1, IL-13Rα2, and IL-4Rα with about a comparable affinity as a wild type IL13.

In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to an IL13 receptor subunit with at least a comparable affinity as a wild type IL13. For example, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 13 receptor alpha 1 (IL-13Rα1), interleukin 13 receptor alpha 2 (IL-13Rα2), interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with at least a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 with at least a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα2 with at least a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-4Rα with at least a comparable affinity as a wild type IL13 sequence. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-13Rα2 with at least a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα with at least a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα2 and IL-4Rα with at least a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1, IL-13Rα2, and IL-4Rα with at least a comparable affinity as a wild type IL13.

In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to an IL13 receptor subunit with at most a comparable affinity as a wild type IL13. For example, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 13 receptor alpha 1 (IL-13Rα1), interleukin 13 receptor alpha 2 (IL-13Rα2), interleukin 4 receptor alpha (IL-4Rα), or a combination thereof with at most a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 with at most a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα2 with at most a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-4Rα with at most a comparable affinity as a wild type IL13 sequence. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-13Rα2 with at most a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1 and IL-4Rα with at most a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα2 and IL-4Rα with at most a comparable affinity as a wild type IL13. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can bind to IL-13Rα1, IL-13Rα2, and IL-4Rα with at most a comparable affinity as a wild type IL13.

In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof can bind to an IL-13Rα1 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL13 sequence. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof can bind to an IL-13Rα1 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL13 sequence.

In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof can bind to an IL-13Rα2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL13 sequence. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof can bind to an IL-13Rα2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL13 sequence.

In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof can bind to an IL-4Rα with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL13 sequence. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof can bind to an IL-4Rα with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL13 sequence.

In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can activate a native IL13 receptor. A native IL13 receptor can be, for example, a receptor comprising an IL-13Rα1 subunit and an IL-4Rα subunit. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can activate a native IL13 receptor when present in a fusion protein. In some embodiments, an IL13 or IL13 variant, derivative, or fragment thereof of the disclosure can activate a native IL13 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native IL13 receptor when present in a fusion protein.

Interleukin 27:

A compound (e.g., polypeptide construct, fusion protein) may comprise an IL27 protein, or a variant, derivative, or fragment thereof operably connected or directly or indirectly fused to an IL4 protein, or a variant, derivative or fragment thereof. The IL27 protein can be a mammalian IL27 protein, such as a human IL27, or mouse IL27, or a variant, derivative, or fragment thereof. An IL27 or an IL27 variant, derivative, or fragment thereof of the disclosure can comprise an IL27A subunit, an IL27B (EBI3) subunit, or a combination thereof. In some embodiments, an IL27 of the disclosure comprises an IL27A subunit. In some embodiments, an IL27 of the disclosure comprises an IL27B subunit. In some embodiments, an IL27 of the disclosure comprises an IL27A subunit and an IL27B subunit. In some embodiments, an IL27 of the disclosure comprises a variant IL27A subunit as disclosed below (e.g., as provided in SEQ ID NO: 24).

TABLE 4 non-limiting examples of IL27 sequences of the disclosure SEQ ID NO: SEQUENCE 24 FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLP LGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERM QLWAMRLDLRDLQRHLRFQVLAAGFNCPEEEEEEEEEEEEERKGLLPGALGSA LQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQ P 25 FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLP LGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERM QLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSA LQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQ P 52 RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARG HSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKP DPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGP IEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK

An example of an amino acid sequence representing IL27A is set forth in SEQ ID NO: 25. An example of an amino acid sequence representing IL27B is set forth in SEQ ID NO: 52. Variants of IL27 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO: 25 or SEQ ID NO: 52, for example, over the entire length. Amino acid sequence identity can be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as disclosed herein. Variants, derivatives, and fragments thereof also include proteins having IL27 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 52. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL27 of the disclosure (e.g., an IL27 variant, derivative, or fragment thereof) can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild type IL27 sequence).

In some embodiments, an IL27 of the disclosure (e.g., an IL27 variant, derivative, or fragment thereof) can comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild type IL27 sequence).

In some embodiments, an IL27 sequence of the disclosure (e.g., an IL27 variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild type IL27 sequence).

In some embodiments, an IL27 sequence of the disclosure (e.g., an IL27 variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to an IL27 sequence disclosed herein (e.g., a wild type IL27 sequence). An amino acid substitution can be a conservative or a non-conservative substitution. The one or more amino acid substitutions, deletions, or insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions can be contiguous, non-contiguous, or a combination thereof.

An IL27 of the disclosure can comprise a wild type IL27 sequence. A non-limiting example of a wild type IL27A sequences is SEQ ID NO: 25, and a non-limiting example of a wild type IL27B sequence is SEQ ID NO: 52. SEQ ID NO: 25 can be a canonical wild type IL27 sequence. In some embodiments, an IL27 sequence of the disclosure comprises one substitution relative to a wild type IL27 sequence.

An example of an IL27 variant, derivative, or fragment thereof of the disclosure is an IL27 variant sequence that can be secreted as a functional immune modulatory monomer protein, for example, an IL27A subunit variant, derivative, or fragment thereof that can be secreted and function as a functional immune modulatory monomer protein without needing to associate with an IL27B (EBI3) subunit. One or more amino acid substitutions, deletions, or insertions can be introduced to generate such a molecule. SEQ ID NO: 24 is an example of an IL27 variant, derivative, or fragment thereof of the disclosure that comprises one amino acid substation (L134C) relative to SEQ ID NO: 25 (which is L162C in the sequence that includes the signal peptide), and can be secreted as a functional immune modulatory monomer protein.

An IL27 of the disclosure can comprise an IL27 variant, derivative, or fragment thereof with one or more amino acid substitutions. For example, an IL27 variant, derivative, or fragment thereof can comprise an amino acid substitution at position F132, N132, L134, P135, E136, E137, L152, L153, P154, or a combination thereof of SEQ ID NO: 25. In some embodiments, an IL27 variant, derivative, or fragment thereof comprises a substitution that is F132C, N132C, L134C, P135C, E136C, E137C, L152C, L153C, P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D, P154D, F132E, N132E, L134E, P135E, E136E, E137E, L152E, L153E, P154E, F132R, N132R, L134R, P135R, E136R, E137R, L152R, L153R, P154R, F132K, N132K, L134K, P135K, E136K, E137K, L152K, L153K, P154K, S31A, L91P, or a combination thereof relative to SEQ ID NO: 25.

In some embodiments, an IL27 variant, derivative, or fragment thereof does not contain a substitution at position F132, N132, L134, P135, E136, E137, L152, L153, or P154 relative to SEQ ID NO: 25. In some embodiments, an IL27 variant, derivative, or fragment thereof does not contain an F132C, N132C, L134C, P135C, E136C, E137C, L152C, L153C, P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D, P154D, F132E, N132E, L134E, P135E, E136E, E137E, L152E, L153E, P154E, F132R, N132R, L134R, P135R, E136R, E137R, L152R, L153R, P154R, F132K, N132K, L134K, P135K, E136K, E137K, L152K, L153K, P154K, S31A, or L91P substitution.

In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure binds to an IL27 receptor subunit with about a comparable affinity as a wild type IL27 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold increased affinity compared to a wild type IL27 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold decreased affinity compared to a wild type IL27 sequence.

For example, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an interleukin 27 receptor alpha (IL-27RA), gp130, or a combination thereof with about a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to IL-27RA with about a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to GP130 with about a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to IL-27RA and GP130 with about a comparable affinity as a wild type IL27.

In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an IL27 receptor subunit with at least a comparable affinity as a wild type IL27. For example, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to IL-27RA, GP130, or a combination thereof with at least a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an IL-27RA with at least a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an GP130 with at least a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an IL-27RA and an GP130 with at least a comparable affinity as a wild type IL27.

In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an IL27 receptor subunit with at most a comparable affinity as a wild type IL27. For example, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to IL-27RA, GP130, or a combination thereof with at most a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an IL-27RA with at most a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an GP130 with at most a comparable affinity as a wild type IL27. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can bind to an IL-27RA and an GP130 with at most a comparable affinity as a wild type IL27.

In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof can bind to an IL-27RA with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL27 sequence. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof can bind to an IL-27RA with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL27 sequence.

In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof can bind to GP130 A with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL27 sequence. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof can bind to GP130 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL27 sequence.

In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can activate a native IL27 receptor. A native IL27 receptor can be, for example, a receptor comprising an IL-27RA subunit and an GP130 subunit. In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can activate a native IL27 receptor when present in a compound of the disclosure (e.g., fusion protein). In some embodiments, an IL27 or IL27 variant, derivative, or fragment thereof of the disclosure can activate a native IL27 receptor when present as a polypeptide that is not part of a compound of the disclosure (e.g., fusion protein), but does not activate native IL27 receptor when present in a compound of the disclosure (e.g., fusion protein).

Interleukin 33:

A compound (e.g., polypeptide construct, fusion protein) may comprise an IL33 protein, or a variant, derivative, or fragment thereof operably connected or directly or indirectly fused to an IL4 protein, or a variant, derivative or fragment thereof. The IL33 protein can be a mammalian IL33 protein, such as a human IL33, or mouse IL33. Non-limiting examples of amino acid sequences representing IL33 include SEQ ID NOs: 26-32.

TABLE 5 non-limiting examples of human IL33 sequences of the disclosure SEQ ID NO: SEQUENCE 26 MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIK KEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRAL HDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYE SQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFF VLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET 27 AFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKK DEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVEL HKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLC TENILFKLSET 28 SGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEK KDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKC EKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENI LFKLSET 29 HDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYE SQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFF VLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET 30 MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIK KEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRAL HDSSITDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSV ELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENL CTENILFKLSET 31 MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIK KEACYFRRETTKRPSLKTGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKD EKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELH KCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTE NILFKLSET 32 MKPKMKYSTNKISTAKWKNTASKALCFKLGNKVLLSYYESQHPSNESGDGVDG KMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFEC KTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET

Variants of IL33 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to any one of SEQ ID NOs: 26-32, for example, over the entire length. Amino acid sequence identity can be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as disclosed herein. Variants, derivatives, and fragments thereof also include proteins having IL33 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of any one of SEQ ID NOs: 26-32. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, an IL33 of the disclosure (e.g., an IL33 variant, derivative, or fragment thereof) can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions relative to an IL33 sequence disclosed herein (e.g., a wild type IL33 sequence).

In some embodiments, an IL33 of the disclosure (e.g., an IL33 variant, derivative, or fragment thereof) can comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to an IL33 sequence disclosed herein (e.g., a wild type IL33 sequence). SEQ ID NO: 26 can be a canonical wild type IL33 sequence.

In some embodiments, an IL33 sequence of the disclosure (e.g., an IL33 variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions relative to an IL33 sequence disclosed herein (e.g., a wild type IL33 sequence).

In some embodiments, an IL33 sequence of the disclosure (e.g., an IL33 variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to an IL33 sequence disclosed herein (e.g., a wild type IL33 sequence). An amino acid substitution can be a conservative or a non-conservative substitution. The one or more amino acid substitutions, deletions, or insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions can be contiguous, non-contiguous, or a combination thereof.

An IL33 of the disclosure can comprise a wild type IL33 sequence. Non-limiting examples of wild type IL33 sequences include SEQ ID NOs: 26-32. SEQ ID NO: 26 can be a canonical wild type IL33 sequence.

An IL33 of the disclosure can comprise an IL33 variant, derivative, or fragment thereof with one or more amino acid substitutions. For example, an IL33 variant, derivative, or fragment thereof can comprise an amino acid substitution at position 1263 of SEQ ID NO: 6. In some embodiments, an IL33 variant, derivative, or fragment thereof comprises a substitution that is 1263M relative to SEQ ID NO: 26.

In some embodiments, an IL33 variant, derivative, or fragment thereof does not contain a substitution at position 1263 relative to SEQ ID NO: 26. In some embodiments, an IL33 variant, derivative, or fragment thereof does not contain an 1263M substitution.

In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure binds to an IL33 receptor subunit with about a comparable affinity as a wild type IL33 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold increased affinity compared to a wild type IL33 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold decreased affinity compared to a wild type IL33 sequence.

For example, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to ST2 (IL1RL1), IL1RAP, or a combination thereof with about a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to ST2 with about a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to IL1RAP with about a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to ST2 and IL1RAP with about a comparable affinity as a wild type IL33.

In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an IL33 receptor subunit with at least a comparable affinity as a wild type IL33. For example, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to ST2, IL1RAP, or a combination thereof with at least a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an ST2 with at least a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an IL1RAP with at least a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an ST2 and an IL1RAP with at least a comparable affinity as a wild type IL33.

In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an IL33 receptor subunit with at most a comparable affinity as a wild type IL33. For example, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to ST2, IL1RAP, or a combination thereof with at most a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an ST2 with at most a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an IL1RAP with at most a comparable affinity as a wild type IL33. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can bind to an ST2 and an IL1RAP with at most a comparable affinity as a wild type IL33.

In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof can bind to an ST2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL33 sequence. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof can bind to an ST2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL33 sequence.

In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof can bind to an IL1RAP with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type IL33 sequence. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof can bind to an IL1RAP with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type IL33 sequence.

In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can activate a native IL33 receptor. A native IL33 receptor can be, for example, a receptor comprising an ST2 subunit and an IL1RAP subunit. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can activate a native IL33 receptor when present in a fusion protein. In some embodiments, an IL33 or IL33 variant, derivative, or fragment thereof of the disclosure can activate a native IL33 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native IL33 receptor when present in a fusion protein.

Transforming Growth Factors (TGF):

A compound (e.g., polypeptide construct, fusion protein) may comprise a TGF protein such as a TGF beta protein, or a variant, derivative, or fragment thereof operatively connected or directly or indirectly fused to an IL4 protein or variant, derivative or fragment thereof.

A compound (e.g., polypeptide construct, fusion protein) may comprise a TGFβ1 protein, or a variant, derivative, or fragment thereof operatively connected or directly or indirectly fused to an IL4 protein or variant, derivative or fragment thereof. The TGFβ1 protein can be a mammalian TGFβ1 protein, such as a human TGFβ1, or mouse TGFβ1. Non-limiting examples of amino acid sequences representing TGFβ1 include SEQ ID NOs: 33 and 34.

TABLE 6 non-limiting examples of human TGFβ1 sequences of the disclosure SEQ ID NO: SEQUENCE 33 MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKL RLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRV LMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQH VELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLS AHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSS RHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCP YIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSN MIVRSCKCS 34 ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSL DTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRS CKCS

Variants of TGFβ1 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to SEQ ID NO: 33 or SEQ ID NO: 34, for example, over the entire length. Amino acid sequence identity can be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as disclosed herein. Variants, derivatives, or fragments thereof also include proteins having TGFβ1 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, a TGFβ1 of the disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions relative to a TGFβ1 sequence disclosed herein (e.g., a wild type TGFβ1 sequence).

In some embodiments, a TGFβ1 of the disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) can comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to a TGFβ1 sequence disclosed herein (e.g., a wild type TGFβ1 sequence).

In some embodiments, a TGFβ1 sequence of the disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions relative to a TGFβ1 sequence disclosed herein (e.g., a wild type TGFβ1 sequence).

In some embodiments, a TGFβ1 sequence of the disclosure (e.g., a TGFβ1 variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to a TGFβ1 sequence disclosed herein (e.g., a wild type TGFβ1 sequence). An amino acid substitution can be a conservative or a non-conservative substitution. The one or more amino acid substitutions, deletions, or insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions can be contiguous, non-contiguous, or a combination thereof.

A TGFβ1 of the disclosure can comprise a wild type TGFβ1 sequence. Non-limiting examples of wild type TGFβ1 sequences include SEQ ID NO: 33 and SEQ ID NO: 34. A canonical TGFβ1 sequence can be SEQ ID NO: 34.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure binds to a TGFβ1 receptor subunit with about a comparable affinity as a wild type TGFβ1 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold increased affinity compared to a wild type TGFβ1 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold decreased affinity compared to a wild type TGFβ1 sequence.

For example, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to a transforming growth factor beta receptor 1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), an (ALK-1), an (ALK-2), or a combination thereof with about a comparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR1 with about a comparable affinity as a wild type TGFB1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR2 with about a comparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to ALK-1 with about a comparable affinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to ALK-2 with about a comparable affinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with about a comparable affinity as a wild type TGFβ1 sequence.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to a transforming growth factor beta receptor 1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), an (ALK-1), an (ALK-2), or a combination thereof with at least a comparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR1 with at least a comparable affinity as a wild type TGFB1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR2 with at least a comparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to ALK-1 with at least a comparable affinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to ALK-2 with at least a comparable affinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at least a comparable affinity as a wild type TGFβ1 sequence.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to a transforming growth factor beta receptor 1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), an (ALK-1), an (ALK-2), or a combination thereof with at most a comparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR1 with at most a comparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR2 with at most a comparable affinity as a wild type TGFβ1. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to ALK-1 with at most a comparable affinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to ALK-2 with at most a comparable affinity as a wild type TGFβ1 sequence. In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can bind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at most a comparable affinity as a wild type TGFβ1 sequence.

In some embodiments, an TGFβ1 or TGFβ1 variant, derivative, or fragment thereof can bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type TGFβ1 sequence. In some embodiments, an TGFβ1 or TGFβ1 variant, derivative, or fragment thereof can bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type TGFβ1 sequence.

In some embodiments, a TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can activate a native TGFβ1 receptor. A native TGFβ1 receptor can be, for example, a receptor comprising a TGFβR1 subunit and a TGFβR2 subunit. In some embodiments, an TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can activate a native TGFβ1 receptor when present in a fusion protein. In some embodiments, an TGFβ1 or TGFβ1 variant, derivative, or fragment thereof of the disclosure can activate a native TGFβ1 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native TGFβ1 receptor when present in a fusion protein.

A compound (e.g., polypeptide construct, fusion protein) may comprise a TGFβ2 protein, or a variant, derivative, or fragment thereof operatively connected or directly or indirectly fused to an IL4 protein or variant, derivative or fragment thereof. The TGFβ2 protein can be a mammalian TGFβ2 protein, such as a human TGFβ2, or mouse TGFβ2. Non-limiting examples of amino acid sequences representing TGFβ2 include SEQ ID NOs: 35-37.

TABLE 7 non-limiting examples of human TGFβ2 sequences of the disclosure SEQ ID NO: SEQUENCE 35 MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPED YPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPF FPSETVCPVVTTPSGSVGSLCSRQSQVLCGYLDAIPPTFYRPYFRIVRFDVSAME KNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTR AEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEA RFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRA LDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSS DTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCK CS 36 ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWS SDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCK CS 37 MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPED YPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPF FPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRI ELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGF KISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSG KTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDL GWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQ DLEPLTILYYIGKTPKIEQLSNMIVKSCKCS

Variants of TGFβ2 include, for example, proteins having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid sequence identity to any one of SEQ ID NOs: 35-37, for example, over the entire length. Amino acid sequence identity can be determined by pairwise alignment using the Needleman and Wunsch algorithm and GAP default parameters as disclosed herein. Variants, derivatives, and fragments thereof also include proteins having TGFβ2 activity, which have been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of any one of SEQ ID NOs: 35-37. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions or insertions.

In some embodiments, a TGFβ2 of the disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions relative to a TGFβ2 sequence disclosed herein (e.g., a wild type TGFβ2 sequence).

In some embodiments, a TGFβ2 of the disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) can comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions, deletions, or insertions relative to a TGFβ2 sequence disclosed herein (e.g., a wild type TGFβ2 sequence).

In some embodiments, a TGFβ2 sequence of the disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions relative to a TGFβ2 sequence disclosed herein (e.g., a wild type TGFβ2 sequence).

In some embodiments, a TGFβ2 sequence of the disclosure (e.g., a TGFβ2 variant, derivative, or fragment thereof) can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions relative to a TGFβ2 sequence disclosed herein (e.g., a wild type TGFβ2 sequence). An amino acid substitution can be a conservative or a non-conservative substitution. The one or more amino acid substitutions, deletions, or insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions, deletions, or insertions can be contiguous, non-contiguous, or a combination thereof.

A TGFβ2 of the disclosure can comprise a wild type TGFβ2 sequence. Non-limiting examples of wild type TGFβ2 sequences include SEQ ID NOs: 35-37. SEQ ID NO: 36 can be a canonical wild type TGFβ2 sequence of the disclosure.

A TGFβ2 of the disclosure can comprise an TGFβ2 variant, derivative, or fragment thereof with one or more amino acid substitutions. For example, a TGFβ2 variant, derivative, or fragment thereof can comprise an amino acid substitution at position R18, P36, or a combination thereof of SEQ ID NO: 36. In some embodiments, a TGFβ2 variant, derivative, or fragment thereof comprises a substitution that is R18C, P36H, or a combination thereof relative to SEQ ID NO: 22. In some embodiments, a TGFβ2, fragment, or derivative thereof comprises the substitutions R18C, and P36H relative to SEQ ID NO: 36.

In some embodiments, a TGFβ2 variant, derivative, or fragment thereof does not contain a substitution at position R18, or P36 relative to SEQ ID NO: 36. In some embodiments, a TGFβ2 variant, derivative, or fragment thereof does not contain a R18C or P36H substitution.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure binds to a TGFβ2 receptor subunit with about a comparable affinity as a wild type TGFβ2 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold increased affinity compared to a wild type TGFβ2 sequence. A comparable affinity can be, for example, less than about 10, less than about 5, less than about 2, less than about 1.9, less than about 1.8, less than about 1.7, less than about 1.6, less than about 1.5, less than about 1.4, less than about 1.3, less than about 1.2, or less than about 1.1 fold decreased affinity compared to a wild type TGFβ2 sequence.

For example, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to a transforming growth factor beta receptor 1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), an (ALK-1), an (ALK-2), or a combination thereof with about a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR1 with about a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR2 with about a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to ALK-1 with about a comparable affinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to ALK-2 with about a comparable affinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with about a comparable affinity as a wild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to a transforming growth factor beta receptor 1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), an (ALK-1), an (ALK-2), or a combination thereof with at least a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR1 with at least a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR2 with at least a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to ALK-1 with at least a comparable affinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to ALK-2 with at least a comparable affinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at least a comparable affinity as a wild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to a transforming growth factor beta receptor 1 (TGFβR1), a transforming growth factor beta receptor 2 (TGFβR2), an (ALK-1), an (ALK-2), or a combination thereof with at most a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR1 with at most a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to TGFβR2 with at most a comparable affinity as a wild type TGFβ2. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to ALK-1 with at most a comparable affinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to ALK-2 with at most a comparable affinity as a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can bind to a TGFβR1, TGFβR2, ALK-1, and ALK-2 with at most a comparable affinity as a wild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof can bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased affinity relative to a wild type TGFβ2 sequence. In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof can bind to a TGFβR1, TGFβR2, ALK-1, or ALK-2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased affinity relative to a wild type TGFβ2 sequence.

In some embodiments, a TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can activate a native TGFβ2 receptor. A native TGFβ2 receptor can be, for example, a receptor comprising a TGFβR1 subunit and a TGFβR2 subunit. In some embodiments, an TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can activate a native TGFβ2 receptor when present in a fusion protein. In some embodiments, an TGFβ2 or TGFβ2 variant, derivative, or fragment thereof of the disclosure can activate a native TGFβ2 receptor when present as a polypeptide that is not part of a fusion protein, but does not activate native TGFβ2 receptor when present in a fusion protein.

In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can bind to receptors present on the surface of a cell and form a complex with about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 receptor subunits (e.g., polypeptide chains). In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can bind to receptors present on the surface of a cell and form a complex with at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 receptor subunits (e.g., polypeptide chains). In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can bind to receptors present on the surface of a cell and form a complex with at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 receptor subunits (e.g., polypeptide chains).

Compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure can comprise two or more covalently bound cytokines. For example, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can comprise an IL4 covalently bound to an IL10, IL13, IL27, IL33, TGFβ1, TGFβ2, or IL4.

A compound (e.g., polypeptide construct, fusion protein) of the disclosure can comprise a linker. For example, an IL4 sequence can be joined to a cytokine (e.g., an IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2) by a linker.

A linker can be a peptide. A linker can comprise a linker sequence, for example, a linker peptide sequence. A linker sequence can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 amino acid residues in length. Examples of linker sequences and linker repeat sequences are provided in Table 8.

TABLE 8 Non-limiting examples of linker  sequences of the disclosure SEQ ID NO: SEQUENCE 38 GSGGGGSGT 39 GGGS 40 GGGGS 41 KESGSVSSEQLAQFRSLD 42 EGKSSGSGSESKST 43 GSAGSAAGSGEF 44 EAAAK 45 EAAAR

A linker as described herein can include a flexible or rigid linker. A flexible linker can have a sequence containing stretches of glycine and serine residues. The small size of the glycine and serine residues provides flexibility, and allows for mobility of the connected functional domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, thereby reducing unfavorable interactions between the linker and protein moieties. Flexible linkers can also contain additional amino acids such as threonine and alanine to maintain flexibility, as well as polar amino acids such as lysine and glutamine to improve solubility.

A flexible linker can comprise SEQ ID NO: 38. A flexible linker can comprise repeats of SEQ ID NO: 39 (GGGS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 39. A flexible linker can comprise repeats of SEQ ID NO: 40 (GGGGS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 40. Several other types of flexible linkers, including SEQ ID NO: 41 (KESGSVSSEQLAQFRSLD) and SEQ ID NO: 42 (EGKSSGSGSESKST), can also be used. The SEQ ID NO: 43 (GSAGSAAGSGEF) linker can also be used, in which large hydrophobic residues are minimized to maintain good solubility in aqueous solutions. The length of the flexible linkers can be adjusted to allow for proper folding or to achieve optimal biological activity of the fused proteins.

A rigid linker can have, for example, an alpha helix-structure. An alpha-helical rigid linker can act as a spacer between protein domains. A rigid linker can comprise repeats of SEQ ID NO: 44 (EAAAK), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 44. A rigid linker can comprise repeats of SEQ ID NO: 45 (EAAAR), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO 45. A rigid linker can have a proline-rich sequence, (XP)n, with X designating alanine, lysine, glutamine, or any amino acid. The presence of proline in non-helical linkers can increase stiffness, and allow for effective separation of protein domains.

A linker of the disclosure can include a non-peptide linker, for example, a chemical linker. For example, two amino acid sequences of the disclosure can be connected together by a chemical linker. Each chemical linker of the disclosure can be alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, any of which is optionally substituted. In some embodiments, a chemical linker of the disclosure can be an ester, ether, amide, thioether, or polyethyleneglycol (PEG). In some embodiments, a linker can reverse the order of the amino acids sequence in a compound, for example, so that the amino acid sequences linked by the linked are head-to-head, rather than head-to-tail. Non-limiting examples of such linkers include diesters of dicarboxylic acids, such as oxalyl diester, malonyl diester, succinyl diester, glutaryl diester, adipyl diester, pimetyl diester, fumaryl diester, maleyl diester, phthalyl diester, isophthalyl diester, and terephthalyl diester. Non-limiting examples of such linkers include diamides of dicarboxylic acids, such as oxalyl diamide, malonyl diamide, succinyl diamide, glutaryl diamide, adipyl diamide, pimetyl diamide, fumaryl diamide, maleyl diamide, phthalyl diamide, isophthalyl diamide, and terephthalyl diamide. Non-limiting examples of such linkers include diamides of diamino linkers, such as ethylene diamine, 1,2-di(methylamino)ethane, 1,3-diaminopropane, 1,3-di(methylamino)propane, 1,4-di(methylamino)butane, 1,5-di(methylamino)pentane, 1,6-di(methylamino)hexane, and pipyrizine. Non-limiting examples of optional substituents include hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, ureido groups, epoxy groups, and ester groups.

In some embodiments, an N terminus of a polypeptide disclosed herein is joined to an N terminus of another polypeptide disclosed herein. In some embodiments, a C terminus of a polypeptide disclosed herein is joined to a C terminus of another polypeptide disclosed herein. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure does not contain a linker.

Compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure can comprise two or more non-covalently bound cytokines. For example, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can comprise an IL4 non-covalently bound to an IL10, IL13, IL27, IL33, TGFβ1, TGFβ2, or IL4.

Amino acids can include genetically encoded and non-genetically encoded occurring amino acids. Amino acids can include naturally occurring and non-naturally occurring amino acids. Amino acids can be L forms or D forms. Substitutions can include conservative and/or non-conservative amino acid substitutions. A conservative amino acid change can be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid substitution can be a substitution of one amino acid for another amino acid of similar biochemical properties (e.g., charge, size, and/or hydrophobicity). A non-conservative amino acid substitution can be a substitution of one amino acid for another amino acid with different biochemical properties (e.g., charge, size, and/or hydrophobicity). A conservative amino acid change can be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids can include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg (R). A conservative amino acid change can be an amino acid change from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids can include Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). A conservative amino acid change can be an amino acid change from one acidic amino acid to another acidic amino acid. Acidic amino acids can include Glu (E) and Asp (D). A conservative amino acid change can be an amino acid change from one basic amino acid to another basic amino acid. Basic amino acids can include His (H), Arg (R) and Lys (K). A conservative amino acid change can be an amino acid change from one polar amino acid to another polar amino acid. Polar amino acids can include Asn (N), Gln (Q), Ser (S) and Thr (T). A conservative amino acid change can be an amino acid change from one nonpolar amino acid to another nonpolar amino acid. Nonpolar amino acids can include Leu (L), Val (V), Ile (I), Met (M), Gly (G) and Ala (A). A conservative amino acid change can be an amino acid change from one aromatic amino acid to another aromatic amino acid. Aromatic amino acids can include Phe (F), Tyr (Y) and Trp (W). A conservative amino acid change can be an amino acid change from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids can include Ala (A), Val (V), Leu (L) and Ile (I). In some embodiments, a conservative amino acid substitution is an amino acid change from one amino acid to another amino acid within one of the following groups: Group I: ala, pro, gly, gin, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

The IL4 can be located N-terminal of the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2). The IL4 can be located C-terminal of the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2). In some embodiments, the C terminus of IL4 can be joined to the N terminus of a linker sequence, and the C terminus of the linker sequence can be joined to the N terminus of the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2). In some embodiments, the N terminus of IL4 can be joined to the C terminus of a linker sequence, and the N terminus of the linker sequence can be joined to the C terminus of the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2).

In an embodiment, the compound (e.g., polypeptide construct, fusion protein) consists essentially of IL4 and the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2). In an embodiment, the compound (e.g., polypeptide construct, fusion protein) consists essentially of IL4, the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2), and a linker.

In an embodiment, the compound (e.g., polypeptide construct, fusion protein) consists of IL4 and the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2). In an embodiment, the compound (e.g., polypeptide construct, fusion protein) consists of IL4, the cytokine (e.g., IL10, IL13, IL27, IL33, TGFβ1, or TGFβ2), and a linker.

In some embodiments, a fusion protein of the disclosure is present in, purified into, and/or used in a multimeric form, for example, a dimeric form or a tetrameric form. In some embodiments, a fusion protein of the disclosure is present as, purified into, and/or used as a monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof. A dimer, trimer, tetramer, or multimer can comprise subunits that are covalently or non-covalently bound.

In some embodiments, an IL4/IL13 fusion protein of the disclosure is present as, purified into, and/or used as a monomer. In some embodiments, an IL4/IL13 fusion protein of the disclosure is present as, purified into, and/or used as a dimer. In some embodiments, an IL4/IL13 fusion protein of the disclosure is present as, purified into, and/or used as a trimer. In some embodiments, an IL4/IL13 fusion protein of the disclosure is present as, purified into, and/or used as a tetramer. In some embodiments, an IL4/IL13 fusion protein of the disclosure is present as, purified into, and/or used as a multimer. In some embodiments, an IL4/IL13 fusion protein is present as, purified into, and/or used as a monomer and a dimer. In some embodiments, an IL4/IL13 fusion protein is present as, purified into, and/or used as a monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof.

In some embodiments, an IL4/IL10 fusion protein of the disclosure is present as, purified into, and/or used as monomer. In some embodiments, an IL4/IL10 fusion protein of the disclosure is present as, purified into, and/or used as dimer. In some embodiments, an IL4/IL10 fusion protein of the disclosure is present as, purified into, and/or used as trimer. In some embodiments, an IL4/IL10 fusion protein of the disclosure is present as, purified into, and/or used as tetramer. In some embodiments, an IL4/IL10 fusion protein of the disclosure is present as, purified into, and/or used as multimer. In some embodiments, an IL4/IL10 fusion protein is present as, purified into, and/or used as monomer and a dimer. In some embodiments, an IL4/IL10 fusion protein is present as, purified into, and/or used as monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof.

In some embodiments, an IL4/IL4 fusion protein of the disclosure is present as, purified into, and/or used as monomer. In some embodiments, an IL4/IL4 fusion protein of the disclosure is present as, purified into, and/or used as dimer. In some embodiments, an IL4/IL4 fusion protein of the disclosure is present as, purified into, and/or used as trimer. In some embodiments, an IL4/IL4 fusion protein of the disclosure is present as, purified into, and/or used as tetramer. In some embodiments, an IL4/IL4 fusion protein of the disclosure is present as, purified into, and/or used as multimer. In some embodiments, an IL4/IL4 fusion protein is present as, purified into, and/or used as monomer and a dimer. In some embodiments, an IL4/IL4 fusion protein is present as, purified into, and/or used as monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof.

In some embodiments, an IL4/IL27 fusion protein of the disclosure is present as, purified into, and/or used as monomer. In some embodiments, an IL4/IL27 fusion protein of the disclosure is present as, purified into, and/or used as dimer. In some embodiments, an IL4/IL27 fusion protein of the disclosure is present as, purified into, and/or used as trimer. In some embodiments, an IL4/IL27 fusion protein of the disclosure is present as, purified into, and/or used as tetramer. In some embodiments, an IL4/IL27 fusion protein of the disclosure is present as, purified into, and/or used as multimer. In some embodiments, an IL4/IL27 fusion protein is present as, purified into, and/or used as monomer and a dimer. In some embodiments, an IL4/IL27 fusion protein is present as, purified into, and/or used as monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof.

In some embodiments, an IL4/IL33 fusion protein of the disclosure is present as, purified into, and/or used as monomer. In some embodiments, an IL4/IL33 fusion protein of the disclosure is present as, purified into, and/or used as dimer. In some embodiments, an IL4/IL33 fusion protein of the disclosure is present as, purified into, and/or used as trimer. In some embodiments, an IL4/IL33 fusion protein of the disclosure is present as, purified into, and/or used as tetramer. In some embodiments, an IL4/IL33 fusion protein of the disclosure is present as, purified into, and/or used as multimer. In some embodiments, an IL4/IL33 fusion protein is present as, purified into, and/or used as monomer and a dimer. In some embodiments, an IL4/IL33 fusion protein is present as, purified into, and/or used as monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof.

In some embodiments, an IL4/TGFβ1 fusion protein of the disclosure is present as, purified into, and/or used as monomer. In some embodiments, a IL4/TGFβ1 fusion protein of the disclosure is present as, purified into, and/or used as dimer. In some embodiments, a IL4/TGFβ1 fusion protein of the disclosure is present as, purified into, and/or used as trimer. In some embodiments, a IL4/TGFβ1 fusion protein of the disclosure is present as, purified into, and/or used as tetramer. In some embodiments, a IL4/TGFβ1 fusion protein of the disclosure is present as, purified into, and/or used as multimer. In some embodiments, a IL4/TGFβ1 fusion protein is present as, purified into, and/or used as monomer and a dimer. In some embodiments a IL4/TGFβ1 fusion protein is present as, purified into, and/or used as monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof.

In some embodiments, a IL4/TGFβ2 fusion protein of the disclosure is present as, purified into, and/or used as monomer. In some embodiments, a IL4/TGFβ2 fusion protein of the disclosure is present as, purified into, and/or used as dimer. In some embodiments, a IL4/TGFβ2 fusion protein of the disclosure is present as, purified into, and/or used as trimer. In some embodiments, a IL4/TGFβ2 fusion protein of the disclosure is present as, purified into, and/or used as tetramer. In some embodiments, an IL4/TGFβ2 fusion protein of the disclosure is present as, purified into, and/or used as multimer. In some embodiments, a IL4/TGFβ2 fusion protein is present as, purified into, and/or used as monomer and a dimer. In some embodiments, a IL4/TGFβ2 fusion protein is present as, purified into, and/or used as monomer, a dimer, a trimer, a tetramer, a multimer, or any combination thereof.

Exemplary Constructs:

In some embodiments, the present disclosure relates to a compound comprising:

-   -   a first binding moiety that binds to a first interleukin         receptor, preferably an interleukin 4 receptor (IL4R),         interleukin 10 receptor (IL10R), interleukin 27 receptor         (IL27R), an interleukin 13 receptor (IL13R), IL27R, or to an         IL33 receptor, a TGFβ1 receptor, and a TGFβ2 receptor; and     -   a second binding moiety that binds to a second interleukin         receptor, preferably chosen from the group consisting of an         interleukin 4 receptor (IL4R), interleukin 10 receptor (IL10R),         an interleukin 13 receptor (IL13R), an IL33 receptor, a TGFβ1         receptor, and a TGFβ2 receptor.

For example, the first binding moiety may bind to an interleukin 4 receptor (IL4R). Or, the first binding moiety may bind to an interleukin 10 receptor (IL10R). Alternatively, the first binding moiety may bind to an interleukin 13 receptor (IL13R) or IL27R. Still alternatively, the first binding moiety may bind to an IL33 receptor. Or, the first binding moiety may bind to a TGFβ1 receptor. Further, the first binding moiety may bind to a TGFβ2 receptor.

In some embodiments, the present disclosure relates to a compound comprising:

-   -   a first binding moiety that binds to a first interleukin         receptor, preferably an interleukin 4 receptor (IL4R); and     -   a second binding moiety that binds to a second interleukin         receptor, preferably chosen from the group consisting of an         interleukin 10 receptor (IL10R), and an interleukin 13 receptor         (IL13R).

In some embodiments, preferably, the first binding moiety and/or the second binding moiety is not a (wild-type) interleukin, and/or does not comprise a (wild-type) interleukin-derived amino acid sequence.

It was found that a compound according to the present disclosure is able to induce unique signaling in neurons, glial cells and other target cells, and can in particular be applied as a treatment of chronic pain and other conditions disclosed herein.

The compound according to the present disclosure may be any kind of compound that can cross-link cytokine receptors disclosed herein (e.g., IL4R and IL10R or IL13R). This compound can be, for example, a complex, or a polypeptide, a fusion protein, or more preferably a bispecific antibody, a bivalent single chain antibody, a bispecific double chain antibody, a triabody, or a tetrabody. The term “first” and “second” binding moiety does not refer to their relative orientation in the compound, i.e. the first binding moiety can be N- or C-terminal to the second binding moiety, or any alternative configuration may be used. The term “first” and “second” only serves to correctly refer to the two different binding moieties in the present disclosure.

The dual binding (or multi binding) compound according to the present disclosure can be employed to connect (e.g. cluster or cross-link) an IL4R and an IL10R or an IL13R in vivo, preferably an IL4R and an IL10R or an IL13R on a sensory neuron or on a glial cell.

In an alternative embodiment, the present disclosure provides for a combination comprising:

-   -   a first binding moiety that binds to an IL4R;     -   a second binding moiety that binds to an IL10R or an IL13R,

wherein the first binding moiety has a linker that binds the second binding moiety or

wherein the second binding moiety has a linker that binds the first binding moiety.

In some embodiments, the first binding moiety and/or the second binding moiety is not a (wild-type) interleukin and/or does not comprise a (wild-type) interleukin-derived amino acid sequence. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure does not contain SEQ ID NO: 11. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure does not contain SEQ ID NO: 15. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure does not contain SEQ ID NO: 46.

The above-mentioned combination can also be employed to connect (cluster or cross-link) cytokine receptors (e.g., an IL4R and an IL10R IL13R, IL27R, IL33R, or TGFβ1R, TGFβ2R) in vivo, for example, an IL4R and an IL10R or an IL13R of a sensory neuron or a glial cell. In this embodiment, the first binding moiety and/or second binding moiety may comprise a tag, and the linker of the respective other binding moiety preferably is a polypeptide that binds to the tag.

The compound or combination according to the present disclosure is provided particularly for use as a medicament or for use in a therapeutic treatment, preferably the treatment of (inflammatory) pain, e.g. chronic (inflammatory) pain, or neurodegenerative disease.

The first binding moiety, the second binding moiety, and/or the linker according to the present disclosure may be or encompass for example a polypeptide, either or not containing sequences of IL4 and IL10 or IL13, respectively, or more preferably an immunoglobulin molecule or epitope-binding fragment thereof, in particular Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, or a complementarity determining region (CDR) fragment, a single chain antibody (scFv), or single domain antibody. A binding moiety or linker according to the present disclosure may thus be or encompass an intact immunoglobulin molecule such as a polyclonal or monoclonal antibody.

A monoclonal (full) antibody comprises two light chains and two heavy chains, each comprising three CDRs, and has a total of 12 CDRs. A CDR region is a variable sequence involved in the physical binding of the antibody to its antigen.

It is possible to select CDR sequences from other species, e.g. murine, and exchange these with CDR sequences in a human immunoglobulin molecule, to obtain a human immunoglobulin molecule having the specificity that is derived from the other species. This may be advantageous as a human sequence may be less immunogenic to humans as compared to the original framework sequence. Such an exchange of CDR sequences is known as humanization. Hence, the compound, immunoglobulin molecule, or first and/or second binding moiety as provided by the disclosure may be humanized.

The first binding moiety, second binding moiety and/or linker according to the present disclosure preferably specifically binds to its respective target. With the term “specifically bind(s) to” is meant that the binding moiety or linker has more affinity towards its target (e.g. human IL4R or human IL10R) or human IL13R than to other molecules present in the target environment (e.g. the human body). The affinity of an antibody to its antigen is expressed as K_(D) (the equilibrium dissociation constant between the antibody and its antigen). It is preferred that the K_(D) of both antigen binding sites of any of the binding moieties disclosed here is lower than 10⁻⁵ M, more preferably lower than 10⁻⁷ M, more preferably lower than 10⁻⁹ M, more preferably lower than 10⁻¹⁰ M.

The compound, first binding moiety, second binding moiety and/or linker according to the present disclosure may be or encompass a single domain antibody. Single domain antibodies (sdAb, also called Nanobody, or VHH) are well known to the skilled person. Single domain antibodies are antibodies whose complementarity determining regions are part of a single domain polypeptide. Single domain antibodies thus comprise a single CDR1, a single CDR2 and a single CDR3. Examples of single domain antibodies are heavy chain only antibodies, antibodies that do not comprise light chains, single domain antibodies derived from conventional antibodies, and engineered antibodies. Single domain antibodies may be derived from any species including mouse, human, camel, llama, goat, rabbit, and bovine. For example, naturally occurring VHH molecules can be derived from Camelidae species, for example in camel, dromedary, alpaca and guanaco.

Like a whole antibody, a single domain antibody is able to bind selectively to a specific target. Single domain antibodies contain only the variable domain of an immunoglobulin chain having CDR1, CDR2 and CDR3 and framework regions. With a molecular weight of only about 12-15 kDa, single domain antibodies are much smaller than regular antibodies (150-160 kDa) which are composed of at least two heavy chains and two light chains. The format of a single domain antibody has the advantage of less sterical hindering when bound to its target and may bind to epitopes not accessible to regular antibodies. This is both advantageous for the specific blocking and cross-linking properties as aimed for in the present disclosure. One other advantage may be that in the case of an allogenic source, single domain antibodies may be less immunogenic in other species than (full) monoclonal antibodies.

In a further aspect of the present disclosure, the compound, first binding moiety and/or second binding moiety according to the present disclosure may comprise a polypeptide selected from the group consisting of a signal sequence, an affinity tag (e.g., a His-tag), and an antibody Fc fragment. Additionally, and/or alternatively, the compound, first binding moiety and/or second binding moiety according to the present disclosure may comprise one or more chemical modifications selected from the group consisting of glycosylation, sialylation, fucosylation, and pegylation.

Pharmaceutical Compositions:

In a preferred embodiment, the compound or combination according to the present disclosure is comprised in a pharmaceutical composition, preferably with or in a pharmaceutically acceptable carrier.

The pharmaceutical composition may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques (e.g., as described in Remington: The Science and Practice of Pharmacy, 19^(th) Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995).

The term “pharmaceutically acceptable carrier” relates to carriers or excipients, which are inherently nontoxic and nontherapeutic. Examples of such excipients are, but are not limited to, saline, Ringer's solution, dextrose, solution and Hank's solution. Non-aqueous excipients such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.

The pharmaceutical composition may be administered by any suitable routes and mode, but preferably by intraarticular or intrathecal administration for example by injection. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

The pharmaceutical compositions according to the invention may be formulated in accordance with routine procedures for administration of compound or combination by injection into a local compartment such as joint or intrathecal space. The pharmaceutical compositions of the present invention include those suitable for intraarticular or intrathecal administration. Or suitable for administration to any other local compartment in the body.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical composition of the disclosure is contemplated. Preferably, the carrier is suitable for local injection in a joint or in the intrathecal space.

A pharmaceutical composition of the disclosure can be a combination of any pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism.

Pharmaceutical formulations for administration can include aqueous solutions of the active compounds in water soluble form. Suspensions of the active compounds can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. The active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

In practicing the methods of treatment or use provided herein, therapeutically-effective amounts of the compounds described herein are administered in pharmaceutical compositions to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

Pharmaceutical compositions can be formulated using one or more physiologically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising compounds described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, entrapping, or compression processes.

The pharmaceutical compositions can include at least one pharmaceutically-acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form. Pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives.

Methods for the preparation of compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically-acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, dispersible granules, and cachets. Liquid compositions include, for example, solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, for example, gels, suspensions and creams. The compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives.

Non-limiting examples of dosage forms suitable for use in the disclosure include liquid, powder, gel, nanosuspension, nanoparticle, microgel, aqueous or oily suspensions, emulsion, and any combination thereof.

Non-limiting examples of pharmaceutically-acceptable excipients suitable for use in the disclosure include binding agents, disintegrating agents, anti-adherents, anti-static agents, surfactants, anti-oxidants, coating agents, coloring agents, plasticizers, preservatives, suspending agents, emulsifying agents, anti-microbial agents, spheronization agents, and any combination thereof.

Non-limiting examples of pharmaceutically-acceptable carriers include saline, Ringer's solution, and dextrose solution. In some embodiments, the pH of the solution can be from about 5 to about 8, and can be from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the compound. The matrices can be in the form of shaped articles, for example, films, liposomes, microparticles, or microcapsules.

The pH of the disclosed composition can range from about 3 to about 12. The pH of the composition can be, for example, from about 3 to about 4, from about 4 to about 5, from about 5 to about 6, from about 6 to about 7, from about 7 to about 8, from about 8 to about 9, from about 9 to about 10, from about 10 to about 11, or from about 11 to about 12 pH units. The pH of the composition can be, for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. The pH of the composition can be, for example, at least 3, at least 4, at least 5, at least 6, at least 6.2 at least 6.4, at least 6.6, at least 6.8, at least 7, at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least 8, at least 9, at least 10, at least 11 or at least 12 pH units. The pH of the composition can be, for example, at most 3, at most 4, at most 5, at most 6, at most 6.2 at most 6.4, at most 6.6, at most 6.8, at most 7, at most 7.2, at most 7.4, at most 7.6, at most 7.8, at most 8, at most 9, at most 10, at most 11, or at most 12 pH units. A pharmaceutical formulation disclosed herein can have a pH of from about 5.5 to about 8.5.

Formulations of the disclosure can comprise sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. These compositions can also contain preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds can be achieved by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. If desired the formulation can be diluted prior to use with, for example, an isotonic saline solution or a dextrose solution.

In some embodiments, the pharmaceutical composition provided herein comprises a therapeutically effective amount of a compound (e.g., polypeptide construct) in admixture with a pharmaceutically-acceptable carrier and/or excipient, for example, saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives, and other proteins. Illustrative agents include octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol.

In some embodiments, a pharmaceutical formulation disclosed herein can comprise: (i) a compound or polypeptide construct disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical formulation.

For solid compositions, solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

A pharmaceutical carrier or excipient can be a solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that is physiologically compatible. The carrier can be suitable for administration by a route disclosed herein (e.g., parenteral).

A composition of the invention can be, for example, an immediate release form or a controlled release formulation. An immediate release formulation can be formulated to allow the compounds to act rapidly. Non-limiting examples of immediate release formulations include readily dissolvable formulations. A controlled release formulation can be a pharmaceutical formulation that has been adapted such that release rates and release profiles of the active agent can be matched to physiological and chronotherapeutic requirements or, alternatively, has been formulated to effect release of an active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through), granules within a matrix, polymeric mixtures, and granular masses.

In some embodiments, a formulation of the disclosure contains a thermal stabilizer, such as a sugar or sugar alcohol, for example, sucrose, sorbitol, glycerol, trehalose, or mannitol, or any combination thereof. In some embodiments, the stabilizer is a sugar. In some embodiments, the sugar is sucrose, mannitol or trehalose.

Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

A pharmaceutical composition can be administered in a local manner, for example, via injection of the compound directly into an organ, optionally in a depot or sustained release formulation or implant. A pharmaceutical composition can be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. A rapid release form can provide an immediate release. An extended release formulation can provide a controlled release or a sustained delayed release.

In some embodiments, a pump can be used for delivery of the pharmaceutical composition. In some embodiments, a pen delivery device can be used, for example, for subcutaneous delivery of a composition of the disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device can use a replaceable cartridge that contains a pharmaceutical composition disclosed herein. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. A disposable pen has no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

A pharmaceutical composition described herein can be in a unit dosage form suitable for a single administration of a precise dosage. In unit dosage form, the formulation can be divided into unit doses containing appropriate quantities of one or more compounds, polypeptide constructs, and/or therapeutic agents. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged injectables, vials, and ampoules. An aqueous suspension composition disclosed herein can be packaged in a single-dose non-reclosable container. Multiple-dose reclosable containers can be used, for example, in combination with or without a preservative. A formulation for injection disclosed herein can be present in a unit dosage form, for example, in ampoules, or in multi dose containers with a preservative.

In some embodiments, a pharmaceutical formulation disclosed herein is a liquid formulation that can comprise about 50 μg/mL to about 100 mg/mL of polypeptide construct. A formulation can comprise, for example, at least 50 μg/mL, at least 100 μg/mL, at least 200 μg/mL, at least 300 μg/mL, at least 400 μg/mL, at least 500 μg/mL, at least 600 μg/mL, at least 700 μg/mL, at least 800 μg/mL, at least 900 μg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 20 mg/mL, or at least 50 mg/mL. In some embodiments, a formulation can comprise at most 100 μg/mL, at most 200 μg/mL, at most 300 μg/mL, at most 400 μg/mL, at most 500 μg/mL, at most 600 μg/mL, at most 700 μg/mL, at most 800 μg/mL, at most 900 μg/mL, at most 1 mg/mL, at most 10 mg/mL, at most 20 mg/mL, or at most 50 mg/mL.

Actual dosage levels of the compound according to the present disclosure may be varied so as to obtain an amount which is effective (“effective amount”) to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compositions of the present invention employed, the route and time of administration, the duration of the treatment, age, sex, weight, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

In one embodiment, the compound or combination of the present disclosure can be given as a bolus injection, in another embodiment, they can be administered by slow continuous administration via an intrathecal pump or the like, over a long period, such as more than 24 hours, in order to reduce toxic side effects.

In yet another embodiment, the compound or combination of the present disclosure can be administered as maintenance therapy, for a period of up to 6 months or more by repeated injections such as, e.g., once a week or 1-10 times per month, for example in a joint or the intrathecal space, or via a pump or any other intrathecal or intraarticular delivery device.

Therapeutic agents described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic agent can vary. For example, the compositions can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The compositions can be administered to a subject during or as soon as possible after the onset of the symptoms. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein. A therapeutic agent can be administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. The length of treatment can vary for each subject.

A dose can be based on the amount of the compound (e.g., polypeptide construct, fusion protein) per kilogram of body weight of a subject. A dose a of a compound can be at least about 0.5 μg/kg, 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μ μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 pg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, or 1 mg/kg. A dose a of a compound (e.g., polypeptide construct, fusion protein) can be at most about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μ μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, or 1 mg/kg.

In some embodiments, a dose can be at least about 1 ng, at least 10 ng, at least 100 ng, at least 500 ng, at least at least 1 μg, at least 5 μg, at least at least 10 μg, at least 50 μg, at least at least 100 μg, at least 500 μg, at least at least 1 mg, at least 5 mg, at least 10 mg, at least 50 mg, or at least 100 mg. A dose can be at most about 1 ng, at most 10 ng, at most 100 ng, at most 500 ng, at most at most 1 μg, at most 5 μg, at most at most 10 μg, at most 50 μg, at most at most 100 μg, at most 500 μg, at most at most 1 mg, at most 5 mg, at most 10 mg, at most 50 mg, or at most 100 mg.

A dose can be determined by reference to a plasma concentration or a local concentration of the polypeptide construct. A target plasma concentration or local concentration of the compound (e.g., polypeptide construct, fusion protein) can be at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. A target plasma concentration or local concentration of the compound (e.g., polypeptide construct, fusion protein) can be at most about 1 nM, at most about 10 nM, at most about 100 nM, at most about 1 μM, at most about 10 μM, at most about 100 μM, or at most about 1 mM.

Neuronal and Nervous System Modulation:

In some embodiments, the disclosure provides methods that involve contacting a cell, such as a nervous system cell, with a compound (e.g., polypeptide construct, fusion protein) of the disclosure. A nervous system cell can be, for example, a neuron, a central nervous system cell, a peripheral nervous system cell, a neuron, a glial cell, a microglial cell, an astrocyte, a schwann cell, an oligodendrocyte, an infiltrating cell, an infiltrating immune cell, an infiltrating myeloid cell, an infiltrating lymphoid cell, an infiltrating macrophage, an infiltrating neutrophil, an infiltrating lymphocyte, an infiltrating T cell, an infiltrating B cell, or an infiltrating natural killer cell. A neuron can be, for example, a sensory neuron, a somatosensory neuron, a visceral sensory neuron, a nociceptor, and/or an autonomic neuron

Contacting a nervous system cell with a compound (e.g., polypeptide construct, fusion protein) of the disclosure can modulate a signaling pathway in the nervous system cell, for example, compared to a nervous system cell that is contacted with equivalent amounts of the an IL4 that is present in the polypeptide construct, a cytokine that is present in the compound (e.g., IL10, IL13, IL27, IL33, TGFβ1, TGFβ2), or a combination of the IL4 and the cytokine. Non-limiting examples of signaling pathways include those disclosed in examples 5 and 7 and FIGS. 8A-8H and 9E-9F. Non-limiting examples of kinases that can be modulated include those disclosed in example 5, example 7, FIGS. 8A-8H, and FIGS. 10A-10E. Non-limiting examples of phosphorylation substrates that can be modulated include those disclosed in example 5, example 7, and FIGS. 8A-9H. Modulation of a signaling pathway can be as determined by, for example, kinase array profiling, detection of phosphorylated substrates via western blot, detection of phosphorylated substrates via flow cytometry or mass cytometry, detection of phosphorylated substrates via ELISA, or detection of phosphorylated substrates or second messengers via mass spectrometry. The nervous system cell can be contacted with an amount of the compound (e.g., polypeptide construct, fusion protein) that is sufficient to modulate the signaling pathway. The nervous system cell can be contacted with a concentration of the compound that is sufficient to modulate the signaling pathway. A concentration of the compound (e.g., polypeptide construct, fusion protein) that is sufficient to modulate the signaling pathway can be, for example, at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The nervous system cell can be contacted with the compound (e.g., polypeptide construct, fusion protein) for a period of time that is sufficient to modulate the signaling pathway. A period of time sufficient to modulate the signaling pathway can be, for example, at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, or at least about a week.

Modulating the activity of a signaling pathway can comprise increasing the activity of the signaling pathway. Modulating the activity of a signaling pathway can comprise decreasing the activity of the signaling pathway. Modulating the activity of a signaling pathway can comprise increasing the activity of a kinase, group of kinases, or kinase class. Modulating the activity of a signaling pathway can comprise decreasing the activity of a kinase, group of kinases, or kinase class. Modulating the activity of a signaling pathway can comprise increasing the phosphorylation of a substrate, group of substrates, or substrate class. Modulating the activity of a signaling pathway can comprise decreasing the phosphorylation of a substrate, group of substrates, or substrate class.

In some embodiments, increasing the activity of a signaling pathway, kinase, group of kinases, kinase class, or increasing the phosphorylation level of a substrate, group of substrates, or substrate class, can comprise an increase of at least about at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% 90%, at least about or 100%. In some embodiments, increasing the activity of a signaling pathway, kinase, group of kinases, kinase class, or increasing the phosphorylation level of a substrate, group of substrates, or substrate class, can comprise an increase of at least about at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or at least about 10,000-fold. In some embodiments, increasing can comprise increasing from an undetectable level to a detectable level.

In some embodiments, decreasing the activity of a signaling pathway, kinase, group of kinases, kinase class, or decreasing the phosphorylation level of a substrate, group of substrates, or substrate class, can comprise a decrease of at least about at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% 90%, at least about or 100%. In some embodiments, decreasing the activity of a signaling pathway, kinase, group of kinases, kinase class, or decreasing the phosphorylation level of a substrate, group of substrates, or substrate class, can comprise a decrease of at least about at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or at least about 10,000-fold. In some embodiments, decreasing can comprise decreasing from a detectable level to an undetectable level.

Contacting a nervous system cell with a compound (e.g., polypeptide construct, fusion protein) of the disclosure can modulate a kinomic profile in the nervous system cell, for example, compared to a nervous system cell that is contacted with equivalent amounts of the an IL4 that is present in the polypeptide construct, a cytokine that is present in the compound (e.g., IL10, IL13, IL27, IL33, TGFβ1, TGFβ2), or a combination of the IL4 and the cytokine. Modulation of a kinomic profile can be as determined by, for example, kinase array profiling, detection of phosphorylated substrates via mass cytometry, or detection of phosphorylated substrates via mass spectrometry. The nervous system cell can be contacted with an amount of the compound that is sufficient to modulate the kinomic profile. The nervous system cell can be contacted with a concentration of the compound that is sufficient to modulate the kinomic profile. A concentration of the compound that is sufficient to modulate the kinomic profile can be, for example, at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The nervous system cell can be contacted with the compound (e.g., polypeptide construct, fusion protein) for a period of time that is sufficient to modulate the kinomic profile. A period of time sufficient to modulate the kinomic profile can be, for example, at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, or at least about a week.

Contacting a nervous system cell with a compound (e.g., polypeptide construct, fusion protein) of the disclosure can modulate a gene expression in the nervous system cell, for example, compared to a nervous system cell that is contacted with equivalent amounts of the an IL4 that is present in the polypeptide construct, a cytokine that is present in the compound (e.g., IL10, IL13, IL27, IL33, TGFβ1, TGFβ2), or a combination of the IL4 and the cytokine. Non-limiting examples of genes that can be modulated include those disclosed in example 5 and FIGS. 9A-9F. Modulated gene expression can be as determined by, for example, RNA sequencing (e.g., with principal component analysis and/or hierarchical clustering of differentially regulated genes), microarray profiling, gene arrays, and RT-qPCR. The nervous system cell can be contacted with an amount of the compound (e.g., polypeptide construct, fusion protein) that is sufficient to modulate gene expression. The nervous system cell can be contacted with a concentration of the compound that is sufficient to modulate gene expression. A concentration of the compound that is sufficient to modulate gene expression can be, for example, at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The nervous system cell can be contacted with the compound (e.g., polypeptide construct, fusion protein) for a period of time that is sufficient to modulate gene expression. A period of time sufficient to modulate gene expression can be, for example, at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, or at least about a week.

Modulating gene expression in a nervous system cell can comprise increasing expression of a gene or group of genes. Modulating gene expression in a nervous system cell can comprise decreasing expression of a gene or group of genes.

In some embodiments, increasing gene expression can comprise an increase of at least about at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% 90%, at least about or 100%. In some embodiments, increasing gene expression, can comprise an increase of at least about at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or at least about 10,000-fold. In some embodiments, increasing can comprise increasing from an undetectable level to a detectable level.

In some embodiments, decreasing gene expression can comprise a decrease of at least about at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% 90%, at least about or 100%. In some embodiments, decreasing gene expression, can comprise a decrease of at least about at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or at least about 10,000-fold. In some embodiments, decreasing can comprise decreasing from a detectable level to an undetectable level.

Contacting a neuron with a compound (e.g., polypeptide construct, fusion protein) of the disclosure can modulate a sensitivity threshold of a neuronal response to a stimulus, for example, compared to a neuron that is contacted with equivalent amounts of the an IL4 that is present in the polypeptide construct, a cytokine that is present in the compound (e.g., IL10, IL13, IL27, IL33, TGFβ1, TGFβ2), or a combination of the IL4 and the cytokine. A sensitivity threshold can comprise an amount (e.g., concentration) of a stimulus required to elicit a pre-determined neuronal response, for example, calcium flux above a certain level, depolarization, alteration of depolarization frequency above or below a certain threshold, altered expression, production, and/or release of a neurotransmitter or neuropeptide above or below a certain level, or altered expression/production of a gene to above or below a certain level. A stimulus can be, for, example, capsaicin, a pro-inflammatory mediator, an anti-inflammatory mediator, a drug, a toxin, a toxicant, a chemical stimulus, a mechanical stimulus, a thermal stimulus, or neuronal damage. In some embodiments, a stimulus is not a pro-inflammatory mediator, but the sensitivity threshold is modulated in the presence of a pro-inflammatory mediator. Modulation of a sensitivity threshold can be as determined by measuring a neuronal response as disclosed herein (e.g., above) to a stimulus as disclosed herein (e.g., above). The neuron can be contacted with an amount of the compound (e.g., polypeptide construct, fusion protein) that is sufficient to modulate the sensitivity threshold. The neuron can be contacted with a concentration of the compound that is sufficient to modulate the sensitivity threshold. A concentration of the compound that is sufficient to modulate the sensitivity threshold can be, for example, at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The neuron can be contacted with the compound for a period of time that is sufficient to modulate the sensitivity threshold. A period of time sufficient to modulate the sensitivity threshold can be, for example, at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, or at least about a week.

Contacting a neuron with a compound (e.g., polypeptide construct, fusion protein) of the disclosure can modulate a magnitude of a neuronal response to a stimulus, for example, compared to a neuron that is contacted with equivalent amounts of the an IL4 that is present in the polypeptide construct, a cytokine that is present in the compound (e.g., IL10, IL13, IL27, IL33, TGFβ1, TGFβ2), or a combination of the IL4 and the cytokine. A magnitude of a neuronal response can be an amount of a response quantified upon exposure to a pre-determined stimulus. A neuronal response can be, for example, calcium flux above a certain level, alteration of depolarization frequency above or below a certain threshold, altered expression, production, and/or release of a neurotransmitter or neuropeptide above or below a certain level, or altered expression/production of a gene to above or below a certain level. A stimulus can be, for, example, capsaicin, a pro-inflammatory mediator, an anti-inflammatory mediator, a drug, a toxin, a toxicant, a chemical stimulus, a mechanical stimulus, a thermal stimulus, or neuronal damage. In some embodiments, a stimulus is not a pro-inflammatory mediator, but the magnitude of the neuronal response is modulated in the presence of a pro-inflammatory mediator. Modulation of a magnitude of a neuronal response can be as determined by measuring a neuronal response as disclosed herein (e.g., disclosed above) to a stimulus as disclosed herein (e.g., disclosed above). The neuron can be contacted with an amount of the compound that is sufficient to modulate the magnitude of the neuronal response. The neuron can be contacted with a concentration of the compound that is sufficient to modulate the magnitude of the neuronal response. A concentration of the compound that is sufficient to modulate the magnitude of the neuronal response can be, for example, at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The neuron can be contacted with the compound for a period of time that is sufficient to modulate the magnitude of the neuronal response. A period of time sufficient to modulate the magnitude of the neuronal response can be, for example, at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, or at least about a week.

Contacting a neuron with a compound of the disclosure can modulate a duration of a neuronal response to a stimulus, for example, compared to a neuron that is contacted with equivalent amounts of the an IL4 that is present in the polypeptide construct, a cytokine that is present in the compound (e.g., IL10, IL13, IL27, IL33, TGFβ1, TGFβ2), or a combination of the IL4 and the cytokine. A duration of a neuronal response can be a duration of a response above a threshold upon exposure to a pre-determined stimulus. A neuronal response can be, for example, calcium flux, alteration of depolarization frequency above or below a certain threshold, an altered depolarization threshold, an altered frequency of action potential firing, altered expression, production, and/or release of a neurotransmitter or neuropeptide above or below a certain level, or altered expression/production of a gene to above or below a certain level. A stimulus can be, for, example, capsaicin, a pro-inflammatory mediator, an anti-inflammatory mediator, a drug, a toxin, a toxicant, a chemical stimulus, a mechanical stimulus, a thermal stimulus, or neuronal damage. In some embodiments, a stimulus is not a pro-inflammatory mediator, but the duration of the neuronal response is modulated in the presence of a pro-inflammatory mediator. Modulation of a duration of a neuronal response can be as determined by measuring a duration of a neuronal response as disclosed herein (e.g., disclosed above) to a stimulus as disclosed herein (e.g., disclosed above). The neuron can be contacted with an amount of the compound that is sufficient to modulate the duration of the neuronal response. The neuron can be contacted with a concentration of the compound that is sufficient to modulate the duration of the neuronal response. A concentration of the compound that is sufficient to modulate the duration of the neuronal response can be, for example, at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The neuron can be contacted with the compound for a period of time that is sufficient to modulate the duration of the neuronal response. A period of time sufficient to modulate the duration of the neuronal response can be, for example, at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, or at least about a week.

Contacting a neuron with a compound (e.g., polypeptide construct, fusion protein) of the disclosure can modulate an ectopic neuronal activity, for example, compared to a neuron that is contacted with equivalent amounts of the an IL4 that is present in the polypeptide construct, a cytokine that is present in the compound (e.g., IL10, IL13, IL27, IL33, TGFβ1, TGFβ2), or a combination of the IL4 and the cytokine. An ectopic neuronal activity can comprise, for example, spontaneous afferent neuronal activity, spontaneous sensory afferent action potential activity, spontaneous neuronal depolarization, or a combination thereof. Modulating ectopic neuronal activity can be as determined by, for example, measurement of spontaneous ectopic discharge recorded from peripheral nerve bundles (e.g., of the sciatic nerve in a rat neuropathic pain model), measurement of spontaneous wind=up and after-discharge activity of wide dynamic range dorsal horn neurons recorded from the spinal cord (e.g., in a rat neuropathic pain model), recording ectopic discharge from modified preparations of neuropathic animals, or a multi-well multielectrode array assay using, e.g., using neurons from dorsal root ganglia. The neuron can be contacted with an amount of the compound that is sufficient to modulate the ectopic neuronal activity. The neuron can be contacted with a concentration of the compound that is sufficient to modulate the ectopic neuronal activity. A concentration of the compound that is sufficient to modulate the ectopic neuronal activity can be, for example, at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 10 μM, or at least about 100 μM. The neuron can be contacted with the compound for a period of time that is sufficient to modulate the ectopic neuronal activity. A period of time sufficient to modulate the ectopic neuronal activity can be, for example, at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, or at least about a week.

In some embodiments, decreasing a sensitivity threshold of a neuronal response to a stimulus, a duration of a neuronal response to a stimulus, a magnitude of a neuronal response to a stimulus, or ectopic neuronal activity can comprise a decrease of at least about at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% 90%, at least about or 100%. In some embodiments, decreasing a sensitivity threshold of a neuronal response to a stimulus, a duration of a neuronal response to a stimulus, a magnitude of a neuronal response to a stimulus, or ectopic neuronal activity can comprise a decrease of at least about at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or at least about 10,000-fold. In some embodiments, decreasing can comprise decreasing from a detectable level to an undetectable level.

In some embodiments, a nervous system cell can be contacted with compound (e.g., polypeptide construct, fusion protein) of the disclosure at a concentration that is at most about 1M, at most about 10 nM, at most about 100 nM, at most about 1 μM, at most about 10 μM, at most about 100 μM, or at most about 1 mM.

A stimulus of the disclosure can comprise or can be a pro-inflammatory mediator. In some embodiments, a stimulus is not a pro-inflammatory mediator, but the sensitivity threshold is modulated in the presence of a pro-inflammatory mediator. Non-limiting examples of pro-inflammatory mediators include a pro-inflammatory cytokine, a pro-inflammatory chemokine, a vasoactive amine, a prostaglandin, a leukotriene, a thromboxane, an oxygen- and/or nitrogen-derived free radical, histamine, a Th1 cytokine, a Th2 cytokine, a Th17 cytokine, IL-1β, APRIL, IFN-α, IFN-β, IFN-γ, IL-1a, IL-1β, IL-2, IL-6, IL-8, IL-9, IL-12, IL-23, LIGHT, TNF-α, and TNF-β. In some embodiments, stimulus does not contain a pathogen associated molecular pattern. In some embodiments, a stimulus does not contain LPS.

In some embodiments, a stimulus comprises a pathogen associated molecular pattern (PAMP) or a damage associated molecular pattern (DAMP). In some embodiments, a signaling pathway, activity of a kinase, kinomic profile, level of phosphorylation of a substrate, expression of a gene, sensitivity of a neuronal response to a stimulus, magnitude of a neuronal response to a stimulus, duration of a neuronal response to a stimulus, ectopic neuronal activity, or a combination thereof is modulated in a presence of a PAMP. In some embodiments, a signaling pathway, activity of a kinase, kinomic profile, level of phosphorylation of a substrate, expression of a gene, sensitivity of a neuronal response to a stimulus, magnitude of a neuronal response to a stimulus, duration of a neuronal response to a stimulus, ectopic neuronal activity, or a combination thereof is modulated in a presence of a DAMP. Non-limiting examples of DAMPs include Biglycan, Decorin, Versican, LMW hyaluronan, Heparan sulfate, Fibronectin (EDA domain), Fibrinogen, Tenascin C, Uric acid, S100 proteins, Heat shock proteins, ATP, F-actin, Cyclophilin A, Aβ, Histones, HMGB1, HMGN1, IL-1a, IL33, SAP130, DNA, RNA, mtDNA, TFAM, Formyl peptide, mROS, Calreticulin, Defensins, Cathelicidin (LL37), EDN, Granulysin, Syndecans, and Glypicans.

Therapeutic Methods:

Compounds (e.g., polypeptide constructs, fusion proteins) disclosed herein can be useful for treating a condition (e.g., disease) in a subject, for example, pain, neuropathy, inflammation, and other conditions.

Pain can include pain that is mediated by the central nervous system, the peripheral nervous system, or a combination thereof. Non-limiting types of pain include, nociceptive pain, peripheral and central neuropathic pain, and mixed types of pain. Neuropathy can contribute to pain. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can be used for treating a neuropathy. A neuropathy can be associated with pain, numbness, weakness, or a combination thereof.

Particularly, it was found that the compound or combination of the present disclosure has a cartilage-protective activity. Therefore, the compound or combination may be used for prevention and treatment of cartilage breakdown, particularly in OA. The compound or combination may be particularly useful for prevention or treatment of OA (prevention or treatment of cartilage degradation) either or not associated with chronic pain.

Moreover, it was also found that the compound or combination of the present disclosure has a neuro-protective activity. Therefore, the compound or combination may be used for prevention and treatment of neuro-degenerative disorders. The compound or combination may be particularly useful for treatment of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or multiple sclerosis.

The compound, combination, or nucleic acid encoding the same according to the present disclosure can be used in therapeutic treatment.

In one embodiment, the compound, combination, or nucleic acid encoding the same is for, or limited to, topical administration or administration to a local compartment of a human or animal body, and/or wherein the compound or combination is for treatment of a (local) part of the human or animal body, for example a knee, hip, joint, or spine. Said local compartment typically has a barrier function that prevents that upon administration more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 wt. % of the compound or combination per day is absorbed into systemic circulation.

Further, the compound or combination according to the present disclosure is particularly suitable for intrathecal or intraarticular administration, for example by injection into an intrathecal space or intraarticular space.

In a further aspect, the present invention pertains to the compound or combination, or a pharmaceutical composition comprising said according to the present disclosure for use in prevention or treatment of (chronic) pain, a condition characterized by local or systemic inflammation, immune activation, neuro-inflammation and/or neurodegeneration. In an embodiment, said condition characterized by local or systemic inflammation, and/or immune activation is selected from the group consisting of: chronic neuropathic, nociceptive, or mixed neuropathic-nociceptive pain, sepsis, adult respiratory distress syndrome, allo- and xenotransplantation, dermatitis, inflammatory bowel disease, sarcoidosis, allergies, psoriasis, ankylosing spondylarthritis, osteoarthritis, autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, glomerulonephritis, immune complex-induced and other forms of vasculitis, Sjogren's disease, gout, burn injuries, multiple trauma, stroke, myocardial infarction, atherosclerosis, diabetes mellitus, extracorporeal dialysis and blood oxygenation, ischemia-reperfusion injuries, and toxicity induced by the in vivo administration of cytokines or other therapeutic monoclonal antibodies.

In a further aspect, the present invention pertains to the compound, combination, or nucleic acid encoding the same according to the present disclosure (e.g. a fusion protein of IL4 and IL10 or IL13 or a bispecific antibody against IL4R and IL10R or IL13R), in a pharmaceutical composition for use in treatment a condition characterized by neuroinflammation or neurodegeneration such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis.

In an embodiment, said condition is characterized by pain and may be selected from pathological pain, visceral or non-visceral inflammatory pain, visceral or non-visceral nociceptive pain, peripheral and/or central neuropathic pain or mixed nociceptive and neuropathic pain.

In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure or nucleic acid encoding the same can be used for treating Alpers' Disease, Arachnoiditis, Arthrofibrosis, Ataxic Cerebral Palsy, Autoimmune Atrophic Gastritis, Amyloidosis, hATTR Amyloidosis, Avascular Necrosis, Back Pain, Batten Disease, Behcet's Disease (Syndrome), Breakthrough Pain, Burning Mouth Syndrome, Bursitis, Central Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (Cadasil), Cerebral ischemia, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Carpal Tunnel, Cauda Equina Syndrome, Central Pain Syndrome, Cerebral Palsy, Cerebrospinal Fluid (CSF) Leaks, Cervical Stenosis, Charcot-Marie-Tooth (CMT) Disease, Chronic Functional Abdominal Pain (CFAP), Chronic Pancreatitis, Collapsed Lung (Pneumothorax), Corticobasal Degeneration, Compression injury, Corneal Neuropathic Pain, Crush syndrome, Degenerative Disc Disease, Dermatomyositis, Dementia, Dystonia, Ehlers-Danlos Syndrome (EDS), Endometriosis, Eosinophilia-Myalgia Syndrome (EMS), Erythromelalgia, Failed Back Surgery Syndrome (FBSS), Fibromyalgia, Friedreich's Ataxia, Frontotemporal dementia, Glossopharyngeal neuralgia, Growing Pains, Herniated disc, Hydrocephalus, Intercostal Neuraligia, Interstitial Cystitis, Juvenile Dermatositis, Knee Injury, Leg Pain, Lewy Body Dementia, Loin Pain-Haematuria Syndrome, Lyme Disease, Meralgia Paresthetica, Mitochondrial Disorders, Mixed dementia, Motor neurone diseases (MND), Monomelic Amyotrophy, Multiple system atrophy (MSA), Myositis, Neck Pain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis, Paget's Disease, Parsonage Turner Syndrome, Pelvic Pain, Peripheral Neuropathy, Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis, Polymyalgia Rhuematica, Polymyositis, Post Herniorraphy Pain Syndrome, Post Mastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy Pain Syndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, Psoriatic Arthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome, Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis, Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Herpes Zoster Shingles, Spasmodic Torticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, Spinal Stenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, Thoracic Outlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, Traumatic Brain Injuries, Vascular Pain, Vulvodynia, Whiplash, or a combination thereof.

In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure or nucleic acid encoding the same can be used to treat an autoimmune disease. Non-limiting examples of autoimmune diseases include Acute disseminated encephalomyelitis, Acute motor axonal neuropathy, Addison's disease, Adiposis dolorosa, Adult-onset Still's disease, Alopecia areata, Ankylosing Spondylitis, Anti-Glomerular Basement Membrane nephritis, Anti-neutrophil cytoplasmic antibody-associated vasculitis, Anti-N-Methyl-D-Aspartate Receptor Encephalitis, Antiphospholipid syndrome, Antisynthetase syndrome, Aplastic anemia, Autoimmune Angioedema, Autoimmune Encephalitis, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune neutropenia, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmune polyendocrine syndrome type 2, Autoimmune polyendocrine syndrome type 3, Autoimmune progesterone dermatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura, Autoimmune thyroiditis, Autoimmune urticaria, Autoimmune uveitis, Balo concentric sclerosis, Behçet's disease, Bickerstaffs encephalitis, Bullous pemphigoid, Celiac disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, Complex regional pain syndrome, CREST syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Discoid lupus erythematosus, Endometriosis, Enthesitis, Enthesitis-related arthritis, Eosinophilic esophagitis, Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, Fibromyalgia, Gastritis, Gestational pemphigoid, Giant cell arteritis, Goodpasture syndrome, Graves' disease, Graves ophthalmopathy, Guillain-Barre syndrome, Hashimoto's Encephalopathy, Hashimoto Thyroiditis, Henoch-Schonlein purpura, Hidradenitis suppurativa, Idiopathic dilated cardiomyopathy, Idiopathic inflammatory demyelinating diseases, IgA nephropathy, IgG4-related systemic disease, Inclusion body myositis, Inflamatory Bowel Disease (IBD), Intermediate uveitis, Interstitial cystitis, Juvenile Arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease, Lupus nephritis, Lupus vasculitis, Lyme disease, Meniere's disease, Microscopic colitis, Microscopic polyangiitis, Mixed connective tissue disease, Mooren's ulcer, Morphea, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myocarditis, Myositis, Neuromyelitis optica, Neuromyotonia, Opsoclonus myoclonus syndrome, Optic neuritis, Ord's thyroiditis, Palindromic rheumatism, Paraneoplastic cerebellar degeneration, Parry Romberg syndrome, Parsonage-Turner syndrome, Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus, Pemphigus vulgaris, Pernicious anemia, Pityriasis lichenoides et varioliformis acuta, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary immunodeficiency, Primary sclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic arthritis, Pure red cell aplasia, Pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, Relapsing polychondritis, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Rheumatoid vasculitis, Sarcoidosis, Schnitzler syndrome, Scleroderma, Sjogren's syndrome, Stiff person syndrome, Subacute bacterial endocarditis, Susac's syndrome, Sydenham chorea, Sympathetic ophthalmia, Systemic Lupus Erythematosus, Systemic scleroderma, Thrombocytopenia, Tolosa-Hunt syndrome, Transverse myelitis, Ulcerative colitis, Undifferentiated connective tissue disease, Urticaria, Urticarial vasculitis, Vasculitis, and Vitiligo.

In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure or nucleic acid encoding the same can be used for treating neuropathy. Non-limiting examples of neuropathy include post-traumatic peripheral neuropathy, post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, chemotherapy-induced neuropathy, polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial neuropathy, predominantly motor neuropathy, predominantly sensory neuropathy, sensory-motor neuropathy, autonomic neuropathy, idiopathic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudenal neuralgia, atypical trigeminal neuralgia, sciatica, brachial plexopathy, or intercostal neuralgia. A neuropathy can be associated with, for example, pain, numbness, weakness, burning, atrophy, tingling, twitching, or a combination thereof.

In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can be used to treat a condition associated with cancer or chemotherapy, for example, chemotherapy-induced neuropathy, chemotherapy-associated pain, chemo brain, cancer-related cognitive impairment, cancer-related cognitive dysfunction. In some embodiments, the condition is associated with a chemotherapy that is being used to treat acute leukemia, astrocytomas, biliary cancer (cholangiocarcinoma), bone cancer, breast cancer, brain stem glioma, bronchioloalveolar cell lung cancer, cancer of the adrenal gland, cancer of the anal region, cancer of the bladder, cancer of the endocrine system, cancer of the esophagus, cancer of the head or neck, cancer of the kidney, cancer of the parathyroid gland, cancer of the penis, cancer of the pleural/peritoneal membranes, cancer of the salivary gland, cancer of the small intestine, cancer of the thyroid gland, cancer of the ureter, cancer of the urethra, carcinoma of the cervix, carcinoma of the endometrium, carcinoma of the fallopian tubes, carcinoma of the renal pelvis, carcinoma of the vagina, carcinoma of the vulva, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, cutaneous melanoma, ependymoma, epidermoid tumors, Ewings sarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, glioma, hematologic malignancies, hepatocellular (liver) carcinoma, hepatoma, Hodgkin's Disease, intraocular melanoma, Kaposi sarcoma, lung cancer, lymphomas, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, muscle cancer, neoplasms of the central nervous system (CNS), neuronal cancer, small cell lung cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, sarcoma of soft tissue, schwanoma, skin cancer, spinal axis tumors, squamous cell carcinomas, stomach cancer, synovial sarcoma, testicular cancer, uterine cancer, or tumors and their metastases, including refractory versions of any of the above cancers, or any combination thereof.

In some embodiments, a compound (e.g., fusion protein, polypeptide construct) is used to treat a condition, wherein the condition is not a cancer.

The present disclosure further provides a gene therapy vector containing nucleotide sequence(s) coding for the compound or combination according to the disclosure, for use in the prevention or treatment of a condition characterized by chronic pain, neuro-inflammation and/or neuro-degeneration.

Preferably said condition is further characterized by pathological pain, visceral or non-visceral inflammatory pain, visceral or non-visceral nociceptive pain, peripheral or central neuropathic pain, or mixed nociceptive-neuropathic pain, neuro-inflammation, and/or neuro-degeneration.

Alternatively, said condition is selected from the group consisting of post-operative orthopedic surgery pain, musculoskeletal pain, irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or post-operative peripheral neuropathy, diabetic peripheral neuropathy, inflammatory peripheral neuropathy, HIV-associated neuropathy, painful peripheral neuropathy, nerve entrapment syndrome, chemotherapy-associated pain, chemotherapy-induced allodynia, complex regional pain syndrome, post-spinal injury pain, post-stroke pain, multiple sclerosis, chronic widespread pain, low back pain, osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia, polymyalgia rheumatica, Alpers' Disease, Arachnoiditis, Arthrofibrosis, Ataxic Cerebral Palsy, Autoimmune Atrophic Gastritis, Amyloidosis, hATTR Amyloidosis, Avascular Necrosis, Back Pain, Batten Disease, Behcet's Disease (Syndrome), Breakthrough Pain, Burning Mouth Syndrome, Bursitis, Central Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (Cadasil), Cerebral ischemia, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Carpal Tunnel, Cauda Equina Syndrome, Central Pain Syndrome, Cerebral Palsy, Cerebrospinal Fluid (CSF) Leaks, Cervical Stenosis, Charcot-Marie-Tooth (CMT) Disease, Chronic Functional Abdominal Pain (CFAP), Chronic Pancreatitis, Collapsed Lung (Pneumothorax), Corticobasal Degeneration, Compression injury, Corneal Neuropathic Pain, Crush syndrome, Degenerative Disc Disease, Dermatomyositis, Dementia, Dystonia, Ehlers-Danlos Syndrome (EDS), Endometriosis, Eosinophilia-Myalgia Syndrome (EMS), Erythromelalgia, Failed Back Surgery Syndrome (FBSS), Fibromyalgia, Friedreich's Ataxia, Frontotemporal dementia, Glossopharyngeal neuralgia, Growing Pains, Herniated disc, Hydrocephalus, Intercostal Neuraligia, Interstitial Cystitis, Juvenile Dermatositis, Knee Injury, Leg Pain, Lewy Body Dementia, Loin Pain-Haematuria Syndrome, Lyme Disease, Meralgia Paresthetica, Mitochondrial Disorders, Mixed dementia, Motor neurone diseases (MND), Monomelic Amyotrophy, Multiple system atrophy (MSA), Myositis, Neck Pain, Occipital Neuralgia, Osteoporosis, Rhabdomyolysis, Paget's Disease, Parsonage Turner Syndrome, Pelvic Pain, Peripheral Neuropathy, Phantom Limb Pain, Pinched Nerve, Plantar Fasciitis, Polymyalgia Rhuematica, Polymyositis, Post Herniorraphy Pain Syndrome, Post Mastectomy Pain Syndrome, Post Stroke Pain, Post Thorocotomy Pain Syndrome, Post-Polio Syndrome, Primary Lateral Sclerosis, Psoriatic Arthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg Syndrome, Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction, Sarcoidosis, Scheuemann's Kyphosis Disease, Sciatica, Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Herpes Zoster Shingles, Spasmodic Torticollis, Sphincter of Oddi Dysfunction, Spinal Cord Injury, Spinal Stenosis, Syringomyelia, Tarlov Cysts, Tethered Cord Syndrome, Thoracic Outlet Syndrome (TOS), TMJ disorders, Transverse Myelitis, Traumatic Brain Injuries, Vascular Pain, Vulvodynia, Whiplash, and any other condition disclosed herein.

According to one embodiment, the compound or combination taught herein can be used for inhibiting production and release of cytokines and other inflammatory mediators by cells, such as macrophages, monocytes, T-lymphocytes, glial cells and other cells. As a result, the compound or combination of the present disclosure can be used for the preparation of a medicament for attenuating inflammatory reactions by inhibiting the release of cytokines and other inflammatory mediators by these cells in vivo. The compound or combination of the present disclosure can be used as stand-alone drug or in combination with other drugs. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure is administered in combination with an analgesic, for example, acetaminophen/paracetamol, a non-steroidal anti-inflammatory drug (NSAID), or an opioid. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can be used in combination with NSAIDs, such as aspirin, ibuprofen, naproxen, celecoxib, ketorolac, or diclofenac. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can be used in combination with specific COX-2 inhibitors, such as celecoxib (Celebrex®), rofecoxib, or etoricoxib. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure can be used in combination with corticosteroids, such as dexamethasone or glucosteroids (e.g., hydrocortisone and prednisone). In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure is administered in combination with an antagonist of a pro-inflammatory cytokine (e.g., and antibody derivative, or other molecule thereof that binds to TNF-α (e.g., adalimumab, etanercept), IL-17 (e.g., secukinumab), IL-23 (e.g., guselkumab, ildrakizumab), or IL-12 and IL-23 (e.g., ustekinumab). In some embodiments, a fusion protein is administered in combination with hyaluronic acid. Multiple therapeutic agents can be administered in any order or simultaneously. In some embodiments, a compound of the invention is administered in combination with, before, or after another drug.

Treatment (prophylactic or therapeutic) may comprise of administering the compound or combination of the present disclosure systemically, intraarticularly, intrathecally, epidurally, or spinally, by injection or by a drug delivery system suitable for local administration. In some embodiments, a pharmaceutical composition of the disclosure can be administered parenterally, for example, by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intracerebroventricular, intracisternal, or intraparenchymal, e.g., injection and infusion. However, other administration routes as set forth above with respect to pharmaceutical compositions comprising the compound or combination recited above may also be employed. The dose and administration regimen may depend on the pharmacodynamic effect aimed at. Typically, the amount of the compound or combination of first and second binding moieties given will be in the range of 0.5 μg to 1 mg per kg of body weight. The dosage can be determined or adjusted by measuring the amount of circulating or local level of the compound or combination upon administration in a biological compartment or space.

Typically, the compound or combination of the present disclosure may be formulated in such vehicles at a concentration of from about 50 μg to about 100 mg per ml.

In an embodiment, the compound or combination according to the present disclosure is biologically active and/or able to signal cells to downregulate the production of at least one inflammatory cytokine or mediator such as IL1β, IL6, IL8, TNFα. Preferably, at least TNFα, IL6, and IL8 are downregulated.

In particular, the compound or combination according to the present disclosure can be biologically active and/or able to signal neurons and other cells in the dorsal root ganglion and posterior horn of the spinal cord to activate unique kinase profiles and express unique sets of genes leading to resistance to the damaging effects of e.g. chemotherapeutic drugs.

In another embodiment, the compound or combination according to the present disclosure is biologically active and/or able to signal neurons to normalize hypersensitivity induced by inflammatory mediators such as proinflammatory cytokines or prostaglandins.

In another embodiment, the compound or combination according to the present disclosure is biologically active and/or able to de-activate glial cells in the dorsal root ganglion, the spinal cord and/or the central nervous system.

Administration of the compound (e.g., polypeptide construct, fusion protein) can continue for as long as clinically necessary. In some embodiments, a compound of the disclosure can be administered for more than 1 day, more than 1 week, more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, more than 12 months, more than 13 months, more than 14 months, more than 15 months, more than 16 months, more than 17 months, more than 18 months, more than 19 months, more than 20 months, more than 21 months, more than 22 months, more than 23 months, or more than 24 months. In some embodiments, a compound of the disclosure is administered for less than 1 week, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, less than 9 months, less than 10 months, less than 11 months, less than 12 months, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, less than 18 months, less than 19 months, less than 20 months, less than 21 months, less than 22 months, less than 23 months, or less than 24 months.

In some embodiments, a compound (e.g., polypeptide construct, fusion protein) can be administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times over a treatment cycle. In some embodiments, a treatment cycle is 7 days, 14 days, 21 days, or 28 days long. In some embodiments, a treatment cycle is 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 11 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months.

In some embodiments, a compound is administered to a subject once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

In a further aspect, the present disclosure relates to the compound or combination, or a pharmaceutical composition comprising said according to the present disclosure for use as a medicament. In an aspect, the present disclosure pertains to the compound or combination, or a pharmaceutical composition comprising the said for use in preventing or treating neuro-inflammation and/or (chronic) pain, for example chronic neuropathic pain experienced during and after chemotherapy, or postherpetic neuralgia, or post-traumatic neuropathic pain, or painful diabetes neuropathy, and other forms of chronic pain, including nociceptive pain and mixed nociceptive and neuropathic pain. The term “chronic” can be defined at persisting for at least 1, 2, 3, 4, 5, 6, 8, 10, or 12 weeks, at least 1, 2, 3, 4, 5, 6, 8, 10, or 12 months, and/or at least 1, 2, 3, 4, or 5 years.

Methods of Making:

The compound or combination of the present disclosure may be prepared by techniques which are routine to the skilled person. For example, it may be prepared using a technique which provides for the production of recombinant proteins by continuous cell lines in culture. For example, the compound or combination of the present invention can be produced in a host cell using a combination of recombinant DNA techniques and gene transfection methods.

For example, to express the compound or combination according to the present disclosure, a nucleic acid molecule encoding the compound or (components of the) combination can be prepared by standard molecular biology techniques. The nucleic acid molecule of the disclosure is preferably operably linked to transcription regulatory sequences such as a promoter, and optionally a 3′ untranslated region. The nucleic acid molecule of the present disclosure may be inserted into a vector, such as an expression vector, such that the genes are operatively linked to transcriptional and translational control sequences. The expression vector and transcription regulatory sequences are selected to be compatible with the expression host cell used. The nucleic acid molecule encoding a compound or (component(s) of the) combination of the present disclosure may be inserted into the expression vector by routine methods.

Additional amino acid sequences may be present at the N- and/or C-terminus of a compound or polypeptide construct (e.g., fusion protein) for example, at any one or both of the first and second binding moieties according to the present disclosure, e.g., to facilitate purification. For example, a poly-histidine-tag, GST-tag, FLAG-tag, CBP tag, HA tag, or Myc tag may be present at the C- or N-terminus to facilitate purification. In some embodiments, an affinity tag is removed from a compound (e.g., polypeptide construct, fusion protein) of the disclosure, e.g., after purification. In some embodiments, a compound (e.g., polypeptide construct, fusion protein) of the disclosure does not contain an affinity tag, (e.g., the compound can be purified by other methods). Alternatively or additionally, the compound, polypeptide construct (e.g., fusion protein) or combination according to the present disclosure may optionally comprise additional protein moieties, such as moieties capable of targeting, e.g., a protein moiety comprising one or more antibody Fc regions. In some embodiments, a compound (e.g., fusion protein, polypeptide construct) comprises an antibody Fc region. In some embodiments, a compound (e.g., fusion protein, polypeptide construct) comprises an extracellular matrix-binding polypeptide.

The compound or combination according to the present disclosure is preferably an isolated (combination of) protein(s) which can be seen as a protein which is no longer in its natural environment, for example in vitro or in a recombinant host cell.

The present disclosure also encompasses a cell (line) expressing the compound or (first and/or second binding moiety of the) combination as disclosed herein, preferably a Chinese hamster ovary (CHO) cell line, which provides a popular mammalian host for large-scale commercial production of compound or combinations as these cells are safe and allow high volumetric yields.

In another aspect, the present disclosure relates to a host cell comprising a nucleic acid sequence of the present disclosure, or a nucleic acid construct or vector comprising a nucleic acid sequence of the present disclosure. The host cell may be any host cell that can transiently or permanently express the compound or (components of the) combination. The host cell is preferably an animal cell or cell line, such as a mammalian cell or cell line.

In one embodiment the compound or (components of the) combination of the present disclosure is expressed in eukaryotic cell, such as mammalian host cell. Preferred mammalian host cells for expressing include CHO cells (including dhfr-CHO cells, described in (Urlaub et al., 1980), used with a DHFR selectable marker, NS/0 myeloma cells, COS cells, HEK293 cells and SP2.0 cells. When recombinant expression vectors comprising nucleic acid sequences encoding the compound or (components of the) combination are introduced into mammalian host cells, they may be produced by culturing the host cells for a period of time sufficient to allow for expression of the compound or (components of the) combination in the host cells or, more preferably, secretion of thereof into the culture medium in which the host cells are grown. The compound or combination of the present disclosure may be recovered from the culture medium in which the host cells are grown and/or may be purified from the culture medium using standard protein purification methods.

Alternatively, the nucleic acid sequences encoding the compound or (components of the) combination of the disclosure can be expressed in other expression systems, such as e.g. algae, as well as insect cells. Furthermore, the compound or combination can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or eggs from hens, or in transgenic plants.

Introduction of the nucleic acid sequence of the present disclosure into a host cell may be carried out by any standard technique known in the art. For expression of the compound or combination of the present disclosure, the expression vector(s) encoding the compound or combination may be transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, lipofectamine transfection, and freeze-dry method transfection, and the like. Cell lines that secrete the compound or combination of the present disclosure can be identified by assaying culture supernatants for the presence of the compound or combination. The preferred screening procedure comprises two sequential steps, the first being identification of cell lines that secrete the compound or combination, the second being determination of the quality of the compound or combination such as the ability thereof to inhibit cytokine production by blood cells stimulated with LPS or other Toll-like receptor agonists, and others.

In an aspect, the present invention is concerned with a method for producing the compound or combination according to the present disclosure, said method comprising the steps of: culturing a host cell of the present invention under conditions permitting the production of the compound or combination; and optionally, recovering the compound or combination. The skilled person will be capable of routinely selecting conditions permitting production of the compound or combination of the present disclosure. Additionally, a person skilled in the art will be capable of recovering the compound or combination produced using routine methods, which include, without limitation, chromatographic methods (including, without limitation, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, metal binding, and the like), immunoprecipitation, HPLC, ultracentrifugation, precipitation and differential solubilisation, and extraction. As said above, recovery or purification of the compound or combination of the present disclosure may be facilitated by adding, for example, a Histidine-tag to the fusion protein.

In some cases, compounds (e.g., polypeptide constructs, fusion proteins) disclosed herein contain two cytokines, C1 and C2, and are referred to in the format C1/C2 or C1-C2. The order in which the cytokines are presented is not limiting and does not necessarily infer the orientation of the cytokines. For example, C1/C2 or C1-C2 can contain cytokine C1 on the C-terminal side of C2 or on the N-terminal side of C2. Similarly, a cytokine referred to as C1/C2 can be the same as a cytokine referred to as C2/C1 unless otherwise specified.

In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

EXAMPLES

To improve the therapeutic potency of regulatory cytokines such as IL4, IL10 and IL13, the present invention discloses that unique signalling of intracellular processes is induced by crosslinking of receptors for these regulatory cytokines and that this unique signalling in cells in the dorsal root ganglia and the dorsal horn of the spinal cord results in an unprecedented analgesic effect.

Moreover, the present invention provides several compounds that can be used to crosslink regulatory cytokine receptors to induce such a unique cell signalling, which can be used as a medicament to treat chronic pain, and neuro-inflammatory and neurodegenerative conditions. These compounds include genetically engineered bispecific antibody that binds to two different receptors for regulatory cytokines, such as the IL4 receptor (IL4R) and the IL10 receptor (IL10R), or the IL4R and the IL13 receptor (IL13R). Such bispecific antibodies have potent analgesic effects—surprisingly they inhibit chronic pain more effectively than the mere sum of the combination of the two cytokine moieties. Moreover, injection of the anti-IL4R-IL10R bispecific antibody or anti-IL4R-IL13R bispecific antibody may completely resolve chronic pain in animal models.

In order to understand how a bispecific antibody against regulatory cytokine receptors exert its unique analgesic and neuroprotective effects, the inventors considered the mechanisms of action of a fusion protein that binds to both IL4R and IL10R and displays potent analgesic properties.

Similarly, they analysed the potency and mechanisms of a fusion protein that binds to both IL4R and IL13R and which has superior analgesic activity compared to the combination of wild-type IL4 and IL10. To that end, the inventors looked at the way in which cross-linking of IL4R and IL10R may transduce signals to the sensory nervous system. The inventors envisage that the superior analgesic effects resulting from cross-linking IL4R and IL10R result from a unique signalling process that has a unique effect on pain resolving pathways in sensory neurons. To confirm the general applicability of cross-linking of regulatory cytokine receptors as a therapeutic approach for chronic pain, neuroinflammation and neuro-degeneration, the inventors also evaluated the effects of cross-linking IL4 and IL13 receptors.

Materials and Methods Animals

All animal experiments are performed in accordance with international guidelines and with prior approval from the University Medical Center Utrecht experimental animal committee. Experiments were conducted using both male and female mice, all of which were between 8-14 weeks old when tested. Observers who performed the behavioral experiments were blind to the mouse genotype and treatment. The following mice were used: wild type (WT) C57BL/6 mice (Envigo, The Netherlands). For generation of nociceptor-specific IL10R knockout mouse strains we used the Cre-loxP system (Sauer B & Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci USA 85(14):5166-5170). Floxed IL10R mice (Pils M C, et al. (2010) Monocytes/macrophages and/or neutrophils are the target of IL-10 in the LPS endotoxemia model. Eur J Immunol 40(2):443-448) were crossed with Na_(v)1.8-Cre mice (Nassar M A, et al. (2004) Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc Natl Acad Sci USA 101(34):12706-12711) in which Cre expression is driven by the Na_(v)1.8 promoter that is expressed in >90% of nociceptors (Shields S D, et al. (2012) Nav1.8 expression is not restricted to nociceptors in mouse peripheral nervous system. Pain 153(10):2017-2030).

Hyperalgesia Models

Mice received an intraplantar injection of 20 μl λ-carrageenan (2% (w/v), Sigma-Aldrich) dissolved in saline solution (NaCl 0.9%) in both hind paws. To induce transient chemotherapy-induced polyneuropathy (CIPN), paclitaxel (2 mg/kg, Cayman Chemical Company) was injected intraperitoneally on days 0 and 2. To induce persistent paclitaxel-induced CIPN paclitaxel (8 mg/kg, Cayman Chemical Company) was injected intraperitoneal on day 0, 2, 4 and 6. To induce persistent oxaliplatin-induced polyneuropathy, mice received two treatment cycles, each consisting of 5 daily intraperitoneal injections of 3 mg/kg oxaliplatin (Tocris) with a 5 days free interval.

To induce transient chemotherapy-induced polyneuropathy (CIPN), paclitaxel (2 mg/kg, Cayman Chemical Company) was injected intraperitoneally on days 0 and 2. To induce persistent paclitaxel-induced CIPN paclitaxel (8 mg/kg, Cayman Chemical Company) was injected intraperitoneal on day 0, 2, 4 and 6. To induce persistent oxaliplatin-induced polyneuropathy, mice received two treatment cycles, each consisting of 5 daily intraperitoneal injections of 3 mg/kg oxaliplatin (Tocris) with a 5 days free interval.

Thermal hyperalgesia was assessed by determining the heat withdrawal latency times using the Hargreaves test (IITC Life Science) (Hargreaves K, Dubner R, Brown F, Flores C, & Joris J (1988) A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32(1):77-88). Mechanical thresholds were determined using the von Frey test with the up-and-down method (Chaplan S R, Bach F W, Pogrel J W, Chung J M, & Yaksh T L (1994) Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53(1):55-63). All experimenters were blind to genotype and/or treatment.

Drugs & Administration

IL4-10 protein was produced in HEK293 cells and purified as described previously (Eijkelkamp N, et al. (2016) IL4-10 Fusion Protein Is a Novel Drug to Treat Persistent Inflammatory Pain. J Neurosci 36(28):7353-7363). Similarly, a fusion protein of IL4 and IL13 (IL4-13) was produced. IL4-10 and IL4-13 fusion protein concentrations were determined based on IL10 ELISA (IL4 Pelipair ELISA kit, Sanquin; IL13 and IL10, DuoSet ELISAs, R&D Systems), as well as on Bicinchoninic Acid Protein Assay (BCA Pierce Protein Assay Kit, ThermoFisher Scientific). Intrathecal (i.t.) injections of different compounds (5 μl/mouse) were performed as described before (Eijkelkamp N, et al. (2010) GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain. J Neurosci 30(6):2138-2149) under light isoflurane/O2 anaesthesia. The IL4-10 fusion protein (1 μg/mouse) or equimolar doses of recombinant human IL4 and IL10 (Sigma) were injected intrathecally at day 6 after intraplantar λ-carrageenan injection.

IL4Rα expression in sensory neurons was knocked down by intrathecal injections of antisense oligodeoxynucleotides (asODN) directed against IL4Rα mRNA (Ripple M J, et al. (2010) Immunomodulation with IL-4R alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease. J Immunol 185:4804-4811). This approach has been shown to successfully inhibit the expression of several proteins in dorsal root ganglia (DRG) neurons (Stone L S & Vulchanova L (2003) The pain of antisense: in vivo application of antisense oligonucleotides for functional genomics in pain and analgesia. Adv Drug Deliv Rev 55(8):1081-1112). AsODN were dissolved in saline (15 μg per 5 μl) and injected intrathecally at day 3, 4 and 5 after intraplantar λ-carrageenan injection. AsODN had a phosphorothioate backbone. The following ODN were used: Mismatch ODN (mmODN): TGGAAAGGCTTATACCCCTC (SEQ ID NO:1); IL4R asODN: CCGCTGTTCTCAGGTGACAT (SEQ ID NO:2).

Tissue Preparation & Immunochemistry

Mice were deeply anaesthetised with an injection of pentobarbital (60 mg/kg; intraperitoneal) and transcardially perfused with Phosphate-buffered saline (PBS; 140 mM NaCl₂, 20 mM Na₂HPO₄, 2.4 mM NaH₂PO₄) followed by 4% paraformaldehyde (PFA) in PBS (Klinipath). Lumbar DRGs and spinal cords (lumbar L3-L5 section) were isolated and postfixed in 4% PFA cryoprotected in sucrose, and embedded and frozen in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura). For IL4R and IL10R stainings, DRGs were directly frozen in OCT without prior fixation. Spinal cords and DRGs were cut in 20 μm and 10 μm thick sections respectively using a cryostat (CM 3050S; Leica). Sections were collected on SuperFrost plus microscope slides (VWR International).

IL4R and IL10R: Sections were fixed with 10% neutral buffer formalin for 10 min and incubated in PBS containing 0.3% Triton X-100 (PBS-T) and 5% normal donkey serum (NDS)). Cultured primary sensory neurons were fixed with 4% PFA for 10 minutes and incubated in PBS containing 0.05% Tween-20, 1% bovine serum albumin (BSA) and 5% NDS). Sections and cells were incubated overnight at 4° C. with rabbit anti-IL4Rα (sc-686, Santa Cruz; 1:100), rabbit anti-IL10Rα (sc-985, Santa Cruz; 1:100), mouse anti-Neurofilament 200 (NO142, Sigma; 1:500), mouse anti-Peripherin (MAB1527, Millipore; 1:100) or Isolectin B4 (B1205, Vector; 1:50) diluted in antibody diluent (PBS-T with 2% BSA). Subsequently, sections were incubated with alexafluor 488- or 594-conjugated secondary antibodies (Thermofisher, 1:1000) followed by DAPI (1:1000, Sigma) staining before sections were mounted on slides with FluorSave reagent (Millipore).

For staining of c-Fos, a marker for neuronal activation, we performed a colorimetric staining as described previously (64) with some modifications. Serial spinal cord sections were incubated in PBS-T containing 0.1% BSA and 2% NDS. Subsequently, sections were incubated with rabbit anti c-Fos (sc-52, Santa Cruz; 1:500) overnight at 4° C. After washing, sections were incubated with Biotin SP conjugated donkey anti-rabbit IgG (1:250, Jackson IR Laboratories) for 90 minutes. Subsequently, sections were incubated with Vector ABC-Elite (1:50, Vector Laboratories) for 90 minutes. Thereafter, sections were developed with diaminobenzidine (DAB)-Nikel solution (0.05M Tris HCl pH 7.6; 3 mg/ml (NH₄)₂Ni(SO₄)₂; 25 mg/ml DAB (1:100, Sigma), hydrogen peroxide (0.1 μl/ml). Finally, the sections were dehydrated and mounted with DePeX (Serva).

Images were taken using a Zeiss Axio Lab A1 (Zeiss). Pictures were analysed with ImageJ (NIH).

Culture of DRG Neurons

DRGs were cultured as described previously (Eijkelkamp N, et al. (2013) A role for Piezo2 in EPAC1-dependent mechanical allodynia. Nat Commun 4:1682). Briefly, DRGs were dissected and placed on ice-cold dissection medium (HBSS w/o Ca²⁺ and Mg²⁺, 5 mM HEPES, and 10 mM glucose). After dissection, axons were cut and dissection medium was replaced by filtered enzyme mix (HBSS w/o Ca²⁺ and Mg²⁺, 5 mM HEPES, 10 mM glucose, 5 mg/ml collagenase type XI (Sigma), and 10 mg/ml Dispase (Gibco)). The DRGs were incubated in enzyme mix for 30 minutes at 37° C. and 5% CO₂. Subsequently, enzyme mix was inactivated with heat-inactivated foetal bovine serum (FBS, Sigma). Cells were cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% FBS (Gibco), 2 mmol/L glutamine (Gibco), 10,000 IU/ml penicillin-streptomycin (Gibco) on poly-L-lysine (0.01 mg/ml, Sigma) and laminin (0.02 mg/ml, Sigma)-coated glass coverslips in a 5% CO₂ incubator at 37° C. Cells were used the following 1-2 days.

To evaluate the effect of cross-linking IL4R, IL10R and IL13R on the damage to neurons induced by chemotherapeutic drugs, cells were incubated in 24 wells plates for 24 hours in presence of paclitaxel (1 uM) or oxaliplatin (5 ug/ml) to induce neurotoxicity. IL4-10 fusion protein (100 ng/mL), IL4-13 fusion protein (100 ng/mL), IL4 an IL13 (50 ng/mL each), IL4 (50 ng/mL), IL10 (50 ng/mL), and IL13 (50 ng/mL) were added together with the chemotherapeutic agent. As controls cells were also cultured in absence of chemotherapeutic drugs or cytokines, and in presence of chemotherapeutic drugs only. After fixation with 4% paraformaldehyde, cells were stained with rabbit anti-mouse βIII-tubulin (ab18207, 1:1000; Abcam). Neurites were visualized with a Zeiss Axio Lab A1 microscope (Zeiss-Oberkochen, Germany) and using a random sampling method, at least 10 images per glass slide were made at a magnification of 10×. The length of neurites was measured with the ImageJ plugin Simple Neurite Tracer76. The averages of neurite length per neuron for a minimum of five neurons per condition were compared between groups for the three individual primary sensory cultures.

Calcium Imaging

DRG neurons used for calcium imaging experiments were stimulated overnight with TNFα (50 ng/ml, Peprotech) with or without the IL4-10 fusion protein (100 ng/ml; 3 nM), recombinant IL4 and IL10 (50 ng/ml each; 3.3 and 2.9 nm, respectively) and/or receptor blocking antibodies targeted against mouse IL4R or IL10R (2 μg/ml, BD pharmingen).

To measure changes in the capsaicin-evoked calcium response, cells were loaded with 5 μM Fura-2-AM (Invitrogen) for 25 minutes in 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM Glucose; pH 7.4. Cells were excited at 340 and 380 nm wavelengths and fluorescence was collected every 3 seconds at 510 nm using an Axio Observer A1 inverted microscope (X20 objective, Zeiss). The ratio 340/380 is directly correlated with the amount of intracellular calcium.

Recordings were performed as previously described (66) with some modifications. Briefly, every experiment included a 5 minutes baseline measurement followed by a stimulation of the cells by superfusion with capsaicin (0.03 μM) for 21 seconds followed by superfusion of medium. A subsequent 5 minutes of superfusion with high K*-buffer (4 mM NaCl, 140 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM Glucose; pH 7.4) was added at the end of each experiment to depolarize the neurons to confirm cell viability and functionality.

RNA Extraction and Quantitative PCR

Lumbar DRGs were homogenized using TRIzol (Invitrogen). Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and 1 μg of total RNA was used to synthesize cDNA. cDNA was synthesized using SuperScript reverse transcriptase (Invitrogen). RNA concentrations were determined using a NanoDrop 2000 (Thermo Scientific).

The real-time PCR reaction using SYBRgreen master mix (BioRad) was performed on an iQ5 Real-Time PCR Detection System (BioRad). Primers used for qPCR are listed in table 9. The mRNA expression levels were normalized for GAPDH, HRPT and actin.

TABLE 9 List of primers used Gene Forward Reverse GAPDH 5′-TGAAGCAGGCATCTGAGGG-3′ 5′-CGAAGGTGGAAGAGTGGGAG- (SEQ ID NO: 3) 3′ (SEQ ID NO: 4) HRPT 5′-TCCTCCTCAGACCGCTTTT-3′ 5′-CCTGGTTCATCATCGCTAATC-3′ (SEQ ID NO: 5) (SEQ ID NO: 6) Actin 5′- 5′-CACTCAGGGCAGGTGAAACT-3′ GATGCACAGTAGGTCTAAGTGGAG- (SEQ ID NO: 8) 3′ (SEQ ID NO: 7) IL4RA 5′-TCTGCATCCCGTTGTTTTGC-3′ 5′-GCACCTGTGCATCCTGAATG-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)

In Situ Proximity Ligation Assay (PLA)

IL4Rα antibody was labelled to thiol-MINUS-oligo with the dual-crosslinker sulfo-SMCC (ThermoFisher). IL10Rα antibody was labelled with biotin using EZ-Link™ Sulfo-NHS-LC-Biotin SMCC (ThermoFischer). The Biotin-PLUS-probe was later bound to the antibody with streptavidin.

Primary DRG cultures were treated with IL4-10 fusion protein (100 ng/ml; 3 nM) or the combination of IL4 and IL10 (50 ng/ml each; 3.3 and 2.9 nM, respectively) for 15 minutes and fixed with 4% PFA for 10 minutes. In situ PLA was performed as described (Soderberg O, et al. (2008) Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45(3):227-232) with some modifications. Samples were blocked with blocking buffer (PBS containing 0.05% Tween-20, 1% BSA and polyA 1:100). Subsequently, samples were incubated overnight at 4° C. with the following antibodies: MINUS probe-labelled rabbit-anti IL4Rα and biotin-labelled rabbit-anti-IL10Rα. For controls biotin-labelled rat anti-CD200 (Serotec) was used instead of anti-IL10Rα antibody. Next, samples were incubated with streptavidin (5 μg/ml) for 25 minutes at room temperature and incubated with biotin linked to a PLUS probe for 30 minutes at room temperature. T4 DNA ligase (5 Weis units/μl) and the circle and linker probes were added (in the presence of 0.1 M DTT and 3 mM ATP) and incubated at 37° C. for 50 minutes in PHI buffer (50 mM Tris-HCl, 10 mM MgCl₂, 10 mM (NH₄)2SO₄, pH 7.5+0.05% Tween20). After washing, samples were incubated with PHI polymerase (kindly donated by Toshiro Kobori), in the presence of 1 mM dNTPs and 0.1 M DTT for 90 minutes at 32° C. Next, samples were stained with antibody against βIII-tubulin, to stain neurons, for 1 hour at RT. After washing, samples were incubated with Cy5-oligonucleotide probe to label amplified DNA and secondary anti-rabbit antibody conjugated with Alexa488. For the minimal (Min) and Maximal (Max) staining control only MINUS probe antibodies were used and the incubation with streptavidin was skipped. For the Max control Biotin-PLUS was added after the ligase incubation. Images were taken using a Zeiss LSM confocal microscope (Zeiss).

Kinase Activity Profiling

Lumbar DRGs were homogenized using M-PER mammalian Extraction buffer (Pierce) supplemented with phosphatase and protease inhibitor cocktails (Pierce). Protein concentration was determined using the Bradford assay (Bio-Rad). Kinase activity profiling was performed using the Tyrosine Kinase PamChip® (PTK) Array and the Serine/Threonine Kinase PamChip® (STK) Array for Pamstation® 12 (PamGene International B.V.). For the PTK arrays 7.5 μg of protein lysate per array were used while for the STK Arrays 2 μg of protein lysate per array were used. Image quantification and statistical analysis were performed using BioNavigator® Software (PamGene International B.V.). Upstream kinase analysis was performed using BioNavigator® Software with peptide-kinase mapping using Kinexus phosphonet enrichment files (www_phosphonet.ca/). Kinome tree illustration was constructed using data from the interactive PamGene BioNaviagor Upstream Kinase Tool (www_kinhub.org/kinmap/index.html). For pathway analyses peptides found to be significantly differentially phosphorylated (p<0.05) between IL4-10 and the combination of the individual cytokines were subjected to pathway analysis using the GeneGo pathway analysis package

RNA Sequencing

RNA libraries were prepared with the poly A selection method followed by multiplexing and sequencing on the Illumina NextSeq500@ platform in a 1×75 bp single-read and 350 million reads per lane (Utrecht Sequencing Facility). All samples passed the read quality checks performed using FastQC (Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. Available online at: www_bioinformatics.babraham.ac.uk/projects/fastqc.). The sequencing reads from each sample were aligned to the recent reference human genome GRCh38 build 79 assembly from Ensembl (Genome Reference Consortium Human Build 38) using STAR aligner (Cunningham F, et al. (2015) Ensembl 2015. Nucleic Acids Res 43(Database issue):D662-669; Anders S, Pyl P T, & Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166-169). Gene expression data for the annotated genes was generated using HTSeq-count (Anders S, Pyl PT, & Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166-169). The samples exhibited batch effect due to different days of isolation and library preparation. The batch effect was corrected using R package RUVSeq (Risso D, Ngai J, Speed T P, & Dudoit S (2014) Normalization of RNA-seq data using factor analysis of control genes or samples. Nat Biotechnol 32(9):896-90). Differential gene expression analysis and variance-stabilizing transformation (to obtain normalized read counts) were performed using R/Bioconductor package DESeq2 (Love M I, Huber W, & Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550).

Results Example 1: Expression of Receptors for Regulatory Cytokines by Sensory Neurons and Glial Cells in the Dorsal Ganglia and Spinal Cord

To identify whether sensory neurons are able to respond to regulatory cytokines, it was analyzed which receptors for regulatory cytokines are expressed by sensory neurons and glial cells. RNAseq data of receptors for IL10, IL4, IL13, and TGFβ1/2 in the dorsal root ganglia and spinal cord were extracted from the data base by Ray et al. (Pain 2018; 159:1325-1345) as available on www_utdallas.edu/bbs/painneurosciencelab/sensoryomics/drgtxome/?go. RNA sequencing revealed expression of receptors for IL10, IL4, IL33, TGFβ1 and TGFβ2 in the dorsal root ganglia and spinal cord of human and mouse (FIG. 1; data are expressed as transcripts per million).

Next, expression of the alpha chain of both cytokine receptors, IL4R (IL4Rα) and IL10R (IL10Rα) in vivo in mice was studied. IL10Rα was expressed in almost all sensory neurons of the DRG, whilst IL4Rα is expressed in subsets of sensory neurons in the DRG (FIG. 2A). In culture, both IL4Rα and IL10Rα are expressed in NF200-, peripherin-expressing neurons and non-peptidergic 1B4+ neurons, often by the same cells.

Example 2: Cross-Linking Regulatory Cytokine Receptors in Dorsal Root Ganglion and Spinal Cord Results in a Potent Analgesic Effect

Intrathecal administration is a well-known method to deliver drugs and other compounds in the direct environment of cells in the dorsal root ganglion and spinal cord. To test the requirement of IL4Rα expression by sensory neurons for the analgesic activity of IL4R-IL10R cross-linking compound, the inventors used intrathecal administration of antisense oligodeoxynucleotides (asODN) targeting IL4Rα mRNA. Three daily intrathecal injections of IL4Rα asODN significantly reduced IL4Rα mRNA and protein expression in the DRG by ˜70% compared to mismatched (mm) ODN-treated animals (FIG. 2B). IL4-10 fusion protein was used to cross-link IL4R and IL10R. Intrathecal injection of IL4-IL10 fusion protein (1 μg) at 6 days after the induction of persistent inflammatory pain completely inhibited carrageenan induced mechanical and thermal hyperalgesia in mice treated with mmODN confirming the potent analgesic properties of crosslinking IL4R and IL10R. It is to be noted that cross linking IL4R and IL10R may not completely abolish ability to detect sensory stimuli, but rather normalizes aberrant pain sensation. IL4Rα knockdown in the DRG markedly reduced the analgesic effect of the IL4R-IL10R cross-linking compound compared to animals treated with mmODN (FIGS. 2C and 2D). Knockdown of IL4Rα during established carrageenan-induced hyperalgesia did not affect the magnitude of mechanical hypersensitivity.

Nav1.8+ sensory neurons mediate inflammatory pain (Abrahamsen et al. Science. 2008 Aug. 1; 321(5889):702-5). To identify the role of IL10Rα expression in the analgesic properties of the IL4R-IL10R cross-linking compound, IL10Rα was selectively ablated in Nav1.8+ neurons (FIG. 2E). The course of carrageenan-induced persistent inflammatory hyperalgesia was indistinguishable between wild-type and Nav1.8-IL10R^(−/−) animals.

Intrathecal injection of the IL4R-IL10R crosslinking compound (1 μg) at day 6 after induction of inflammatory pain attenuated thermal and mechanical hyperalgesia in wild type animals (FIGS. 2F and 2G). In contrast, deletion of IL10Rα in Nav1.8+ nociceptors partially ablated the analgesic effect of the IL4R-IL10R cross-linking compound (FIGS. 2F and 2G), indicating that nociceptor IL10Rα may be required in part for the pain inhibiting effects of IL4R-IL10R cross-linking compound.

Since both receptors are required for full analgesic effect of cross-linking IL4R and IL10R, the inventors next considered if ablation of both receptors in sensory neurons would completely prevent the analgesic actions of the IL4R-IL10R cross-linking compound. To that end, IL4R expression was knocked down in Nav1.8-IL10R−/− mice with intrathecal IL4Rα asODN injections. Knockdown of both IL4Rα and IL10Rα itself does not affect the course of persistent inflammatory pain. Importantly, knockdown of both IL4Rα and IL10Rα expression in the DRGs completely prevented resolution of pain by the IL4R-IL10R cross-linking compound (FIGS. 3A-D).

An increase of spinal immediate-early gene c-Fos expression can be used as a proxy of spinal neuronal activation in the dorsal horn of the spinal cord. At day 7 after intraplantar carrageenan injection the number of c-Fos positive neurons in the superficial layers of the dorsal horn was significantly increased compared to naive animals (FIG. 3C). Intrathecal administration of IL4-10 fusion protein significantly reduces the number of dorsal horn spinal cord c-Fos positive neurons. Knockdown of IL4Rα and IL10Rα in sensory neurons completely prevents IL4R-IL10R cross linking compound mediated attenuation of the c-Fos expression (FIG. 3C). Overall this indicates that both IL4Rα and IL10Rα in sensory neurons are required for the full inhibition of persistent inflammatory pain resulting from cross-linking IL4R and IL10R.

The potential of cross-linking IL4R and IL13R to inhibit pain was investigated in chemotherapy-induced neuropathy models. Mice received 4 injections of paclitaxel (8 mg/kg) every other day from day 0 to 6. Paclitaxel induced mechanical hyperalgesia that started on the first day after the first injection and that persisted at least 3 weeks after chemotherapy-treatment was stopped. Two days after the last paclitaxel injection, mice were injected intrathecally with 3 different doses of IL4-13 fusion protein (0.3, 1 and 3 μg/mouse) to cross-link IL4R and IL13R on cells in the dorsal root ganglion and the spinal cord (FIG. 4A). An almost normalization of mechanical hyperalgesia lasting for at least a week was observed, demonstrating the potential of cross-linking IL4R and IL13R for long-lasting resolution of chemotherapy-induced polyneuropathy. Importantly, cross-linking IL4R and IL13R also reduced paclitaxel-induced intra-epidermal nerve fibre loss in the paw skin (FIG. 4B), demonstrating that this intervention prevents neuronal damage in vivo.

To confirm that ross-linking IL4R and IL13R provides neuroprotection against a broader spectrum of chemotherapy-induced polyneuropathy, toxic neuropathy was also induced in mice using a platinum-based chemotherapeutic drug, oxaliplatin. Two cycles of 5 times a daily injection of oxaliplatin, separated by 5 days without intraperitoneal injection, induced mechanical allodynia that persisted for at least 3 weeks (FIG. 4C). Intrathecal injection of IL4-13 fusion protein on the second day after the last oxaliplatin injection reduced mechanical allodynia significantly for 4 days (FIG. 4C). Intrathecal injection of either wild-type IL4 or wild-type IL13 transiently inhibited oxaliplatin-induced mechanical allodynia for about 1 day, which effect was significantly shorter than that of cross-linking IL4R and IL13R by the fusion protein.

Example 3: Cross-Linking IL4R and IL10R or IL4R and IL13R has Unique Effects on Neurons

Pro-inflammatory mediators sensitize sensory neurons for noxious and innocuous stimuli. To test whether cross-linking of IL4R and IL10R inhibits inflammatory mediator-induced sensitization of capsaicin-induced calcium responses in sensory neurons, cultured neurons were treated with TNF (50 ng/ml) with and without IL4-10 fusion protein, which was used to cross-link IL4R and IL10R (100 ng/ml, 3 nM). TNF significantly increased the magnitude of capsaicin-induced calcium influxes compared to untreated cells (FIG. 5A-C). Co-treatment with IL4-10 fusion protein completely prevents the sensitization of capsaicin-evoked calcium influx by TNFα. In a control experiment, it was excluded that IL4-10 fusion protein affects capsaicin-induced calcium responses in non-sensitized neurons (not shown). Similarly, cross-linking IL4R and IL10R inhibits PGE2-induced sensitization of capsaicin-induced calcium responses (FIG. 5D-F), without affecting normal capsaicin-induced calcium fluxes (not shown). This indicates that cross-linking IL4R and IL10R prevents neuronal sensitization by inflammatory mediators.

To identify whether the inhibitory effects of IL4-10 fusion protein on neuronal sensitization requires both cytokine binding moieties, separate receptor blocking antibodies were added. Blocking either IL4R or IL10R slightly reduced IL4-10 fusion protein-induced inhibition of TNFα-induced sensitization of capsaicin evoked calcium responses. Interestingly, blocking both IL4R and IL10R receptors completely abrogates inhibition of neuronal sensitization induced by IL4-10 fusion protein (FIG. 5G).

Next the inventors considered to what extent cross-linking IL4R and IL10R to normalize neuronal sensitization by inflammatory mediators is superior to those of the combination of the wild-type cytokines. IL4-10 fusion protein dependently inhibits TNFα-induced sensitization of capsaicin-evoked calcium influx. At a concentration of 3 nM, it completely reversed TNFα-induced sensitization, whilst the combination of individual cytokines at the highest dose tested (30 nM) inhibited the TNFα-induced sensitization to a maximum of 50%. This indicates that cross-linking IL4R and IL10R has a superior potency over the combination of wild-type IL4 and IL10 to reduce neuronal sensitization by inflammatory mediators.

Next, the inventors investigated whether such unique effects of crosslinking IL4R and IL10R on neurons also could be found for cross-linking IL4R with IL13R. To evaluate this, mouse sensory neurons were cultured in presence of oxaliplatin with or without IL4-13 fusion to cross-link IL4R and IL13R, or equimolar concentrations of wild-type IL4 and IL13 protein. Neurite length was measured to assess neurotoxicity. Oxaliplatin had a significant negative effect on neurite length when compared to the control group (FIG. 6). IL4-13 fusion protein completely protected against oxaliplatin-induced neurotoxicity, whilst the combination of equimolar concentrations of wild-type IL4 and IL13 did not. An additional assay with two IL4-IL13 fusion proteins showed similar

Thus, these data together demonstrate that cross-linking IL4R and IL10R, or IL4R and IL13R induces effects in neurons that normalize the hypersensitization by inflammatory mediators and protect the cells against damage. Importantly, these effects are unique to cross-linking involved receptors, since they are not induced by combinations of the wild-type cytokines tested at concentrations equimolar to that of the fusion proteins.

Example 4: IL4-10 Fusion Protein Crosslinks IL4R and IL10R in Sensory Neurons

To further investigate the mechanisms of the unique effects of IL4-10 fusion protein and IL4-13 fusion proteins, the inventors decided to analyse the cellular processes induced by IL4-10 fusion protein in more detail. The inventors hypothesized that the IL4-10 fusion protein causes heterologous clustering of IL4R and IL10R, thereby inducing unique signalling in sensory neurons, i.e. signalling that is not induced by wild-type cytokines, and even not by the combination of wild-type cytokines. First, cross-linking of IL4R and IL10R by IL4-10 fusion proteins on neurons was demonstrated. Sensory neurons were incubated IL4-10 fusion protein or the combination of the respective interleukins for 15 minutes in vitro followed by a proximity ligation assay (PLA) to assess clustering of IL4R and IL10R. This method enables the inventors to detect clustering of the 2 receptors within a 51 nm range. IL4-10 fusion protein (100 ng/ml, 3 nM) treatment indeed cluster IL4R and IL10R receptors in sensory neurons, whilst receptor clustering did not occur after treatment with equimolar concentration of the combination of the respective cytokines or after vehicle (FIG. 7). Clustering of IL4R and IL10R is specifically induced by the fusion protein as clustering of IL4R and another highly expressed membrane protein, CD200, was not observed (data not shown).

Example 5: Cross-Linking IL4R and IL10R Induces a Distinct Kinase Activity Profile Compared to the Combination of Wild-Type Cytokines

The ability of IL4-10 fusion protein to cross-link IL4 and IL10 receptors raises the possibility that this drives unique downstream signaling events. To elucidate downstream signaling in sensory neurons in an unbiased manner, the inventors performed PAMgene kinase activity profiling to assess global protein tyrosine kinases (PTK) and serine/threonine kinases (STK) activity in homogenates of lumbar DRGs of mice with persistent inflammatory pain after administration of IL4-10 fusion protein. Kinomic profiles were assessed at 30, 60 and 240 minutes after intrathecal administration with either the anti-IL4R-IL10R bispecific antibody, the combination of cytokines or PBS. Analyses of the 3 different time points indicated that the most prominent changes in kinome profiles were found at 60 minutes after intrathecal injection, whilst differences are less pronounced at the other time points examined (FIG. 8). Analyses of the peptides that are differentially phosphorylated by PTK present in the DRG homogenates at 60 minutes after treatment shows that in total 38 peptides were differentially phosphorylated when in IL4-10 fusion protein-treated mice compared to vehicle treated mice. Cross-linking IL4R and IL10R induces stronger phosphorylation of 5 peptide substrates for PTKs that were also activated by the combination of cytokines (FIG. 8A). Interestingly, 33 peptides are only phosphorylated by homogenates from mice treated with IL4-10 fusion protein and from mice treated with the combination of IL4 and IL10, indicating that anti-IL4R-IL10R bispecific antibody activates a unique set of PTK (FIG. 8A). Peptides were identified that exhibited different phosphorylation between IL4-IL10 treatment and treatment with the combination of unlinked cytokines (FIG. 8B). Next the inventors evaluated the differentially phosphorylated STK peptides. Intriguingly DRG homogenates of mice treated with the combination of the cytokines phosphorylate 14 STK peptides to a lesser extent compared to vehicle-treated mice, whilst the homogenates from mice treated with IL4-10 fusion protein did not exhibit differences for most peptides, and induced increased phosphorylation in some (FIG. 8C). Peptides were identified that exhibited different phosphorylation between IL4-IL10 treatment and treatment with the combination of unlinked cytokines (FIG. 8D). These data indicate that both PTK and STK activity are differentially regulated upon cross-linking of IL4R and IL10R in vivo compared to the effects of the combination of wild-type cytokines. Each of the peptides present in the kinase activity array can be substrates for different kinases, and one kinase or family of kinases can phosphorylate different peptides. To predict the putative upstream kinases activated specifically by cross-linking IL4R and IL10R, the differential phosphorylation profiles are loaded into PhosphoNET. These analyses predict that several kinases are activated at 60 minutes after injection based on the observed phosphorylation patterns of the peptides (FIG. 8E-H). The inventors identified kinases, including the PTK platelet-derived growth factor receptor A (PDGFRa), KIT, fms like tyrosine kinase 3 (FLT3), MER or RET tyrosine kinases; the calcium calmodulin kinases (CAMK) such as CHK2, CAMK4 or DCAMKL2; AMPK, JAK1, and the Protein kinase A/G/C family such as AKT3, PKCb or NDR1/2. Examples of differentially activated kinases between IL4-IL10 and the combination of unlinked cytokines are provided in FIG. 8G and FIG. 8H, and include the PTKs KIT, PDGFRA, FER, MET and JAK1, and the STKs AKT3, LATS2, NDR2 or CAMK2. Pathway analysis of these predicted differentially can activate kinases indicates that the most active cellular processes induced by anti-IL4R-IL10R bispecific antibody treatment are mapped to immune responses, altered transcription, cell adhesion or cell cycle. The top 5 kinase substrates ranked by occurrence in the top 50 of the pathway analysis were NAPDH oxidase P47-phox, p120GAP, Rb protein, CDC25A and ZAP70. Overall this indicates that cross-linking IL4R and IL10R drives different signalling pathways, differential activation of kinases, different phosphorylation of kinase substrates, and a different kinomic profile compared to vehicle and compared to treatment with equivalent amounts of unlinked cytokines. For example, the activation of sets of kinases not activated by the combination of IL4 and IL10 and drives stronger activation of several kinases also activated by the combination of IL4 and IL10.

Example 6: Cross-Linking IL4R and IL10R In Vivo Induces a Unique Transcriptome

To further explore the potential capacity of cross-linking IL4R and IL10R to elicit downstream effects that differ from that of the combination of IL4 and IL10, the inventors performed RNA sequencing of DRGs, six hours after a single intrathecal injection of IL4-10 fusion protein, the combination of IL4 and IL10, or PBS alone in mice with persistent nociceptive pain. Principal component analysis (PCA) indicates that 75% of the variance in gene expression can be explained by the first two principal components. The first principal component captures 55% variance of the data and separates the mice with cross-linking of IL4R and IL10R from mice that received vehicle or the combination of unfused cytokines, while the second principal component may capture 20% variance of the data and separates vehicle treated animals, indicating that IL4-10 fusion protein induces different transcriptome changes (FIG. 9A). Hierarchical clustering of the top 500 differentially regulated genes showed that based on their transcriptional profile the individual animals clustered based on the three different treatments (FIG. 9B). Thus, cross-linking IL4R and IL10R induces a different transcriptional profile compared to mice treated with IL4 and IL10 or vehicle. Treatment of mice with the combination of IL4 and IL10 resulted in 4905 genes differentially expressed (FDR corrected p-value<0.05) compared to those injected with PBS (FIG. 9C). KEGG Pathway analysis indicated that these genes aggregate in pathways affecting TLR signaling, oxidative phosphorylation, ribosomal proteins and lipid metabolism. In the mice injected intrathecally with IL4-10 fusion protein, 3995 genes are differentially expressed (FDR corrected p-value<0.05) compared to those injected with vehicle. Pathway analysis of the differentially expressed genes reveals that neuronal-related genes such as axon guidance and calcium signaling or energy production such as oxidative phosphorylation are downregulated in mice upon cross-linking IL4R and IL10R. Moreover, genes involved in inflammatory pathways like interferon signaling, antigen processing and presentation, complement and interleukin signalling are affected. Interestingly, when genes induced by cross-linking of IL4R and IL10R are compared with those induced by the combination of the cytokines, 3025 genes are differentially expressed (FIG. 9C). Analysis of these genes indicates that the expression of 1650 genes is increased, whilst 1375 are downregulated upon cross-linking IL4R and IL10R, as compared to the combination of cytokines. Importantly, 1675 genes are uniquely regulated by IL4-10 fusion protein. From these uniquely regulated genes, the expression of 981 genes is increased by the fusion protein, whilst 694 are downregulated. Pathway analysis of the genes that are differentially expressed upon crosslinking of IL4R and IL10R as compared to the transcriptome of animals receiving the combination of wild-type IL4 and IL10 indicates that these genes belong to pathways including cytokine signaling, neurotrophin signaling, and pathways affecting innate and adaptative immune system indicating potential DRG-infiltrating immune cells (FIG. 9E). Additional analysis of upstream kinases based on KEGG pathways indicated that signalling pathways including cytokine and chemokine signaling, NOD-like receptor signaling or JAK-STAT signaling were affected (FIG. 9F). Focal and cell adhesion molecules and pathways affecting innate and adaptive immune system, such as TLR, T and B cells receptor pathways were also affected, which may reflect potential DRG-infiltrating immune cells. Overall, RNAseq data demonstrates that cross-linking IL4R and IL10R induces a unique transcriptome change as compared to animals treated with the combination of IL4 and IL10.

Example 7: JAK1 Activation Contributes to the Superior Analgesic Effect of IL4-10

Based on the data obtained from PAMgene analysis, KIT, PDGFRA, FER, MET and JAK1 were identified as the 5 highest ranking potential Tyrosine kinases that could be linked to the superior effects of IL4-10. Whether IL4-10 differentially activates JAK1 in sensory neurons in vitro by was investigated by determining phosphorylation of JAK1 after stimulation of sensory neurons in vitro with IL4-10 or equimolar concentration of IL4 and IL10 for 10, 30 or 60 minutes. After 10 minutes, IL4-10 significantly increased pJAK1 in sensory neurons compared to vehicle and IL4+IL10 (FIG. 10A). At 30 minutes after stimulation both IL4-10 and IL4+IL10 increased pJAK1. To investigate whether JAK1 signaling is required for the superior IL4-10 mediated pain inhibition, carrageenan-injected animals were treated with Ruxolitinib (JAK1/2 inhibitor) for 3 consecutive days, starting one day before intrathecal administration of IL4-10. Ruxolitinib almost completely prevented the IL4-10-induced inhibition of thermal hyperalgesia (FIG. 10B) and shortened the duration of IL4-10-induced inhibition of mechanical (FIG. 10C) hyperalgesia. Inhibition of JAK1 with Ruxolitinob did not affect IL4+IL10-mediated inhibition of hyperalgesia. To verify whether other kinases identified based on PAMgene and RNAseq data (c-Kit, PDGFR or c-Met) also contribute to the pain-inhibiting effects of IL4-10, these were inhibited using Dasatinib (Kit inhibitor), Masitinib (Kit and PDGFR inhibitor) and JNJ-38877605 (c-Met inhibitor). Inhibition of c-Kit or combined inhibition of c-kit and PDGFR did not affect IL4-10-induced inhibition of thermal hyperalgesia (FIG. 10D). In contrast, inhibition of c-Kit and c-Kit and PDGFR enhanced the IL4-10-induced reduction in mechanical hyperalgesia by IL4-10 (FIG. 10E). Pharmacological inhibition of c-Met partially attenuated the pain-inhibiting effects of IL4-10 (FIG. 10 D, E).

These data suggest that pJAK1 and c-MET signaling contribute to the superior pain-alleviating effect of IL4-10.

Example 8: Additional Polypeptide Constructs of the Disclosure

This example demonstrates design and generation of non-limiting examples of IL4-containing compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure.

An IL10/IL4 compound (e.g., polypeptide construct, fusion protein) of the disclosure is designed. SEQ ID NO: 11 is joined to SEQ ID NO: 15 using the SEQ ID NO: 38 linker, resulting in SEQ ID NO: 46. A poly-histidine tag is added to the N-terminus, and the construct is produced by transient transfection of HEK293E cells as disclosed below.

Additional IL10/IL4 compounds (e.g., polypeptide constructs, fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof) is joined to SEQ ID NO: 15 (or a variant, derivative, or fragment thereof), either directly or via a linker as disclosed herein (for example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The compounds (e.g., polypeptide constructs, fusion proteins) are designed in both orientations, e.g., with IL4 located on the C-terminal side of IL10, or with IL4 located on the N-terminal side of IL10. An affinity tag is added to the N-terminus and/or the C-terminus of each construct, and the constructs are produced by transient transfection of HEK293E cells as disclosed below.

An IL13/IL4 compound (e.g., polypeptide construct, fusion protein) of the disclosure is designed. SEQ ID NO: 11 is joined to SEQ ID NO: 22 using the SEQ ID NO: 38 linker, resulting in SEQ ID NO: 47. A poly-histidine tag is added to the N-terminus, and the construct is produced by transient transfection of HEK293E cells as disclosed below.

Additional IL13/IL4 compounds (e.g., polypeptide constructs, fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof) is joined to any one of SEQ ID NOs: 16-23 (or a variant, derivative, or fragment thereof), either directly or via a linker as disclosed herein (for example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The compounds (e.g., polypeptide constructs, fusion proteins) are designed in both orientations, e.g., with IL4 located on the C-terminal side of IL13, or with IL4 located on the N-terminal side of IL13. An affinity tag is added to the N-terminus and/or the C-terminus of each construct, and the constructs are produced by transient transfection of HEK293E cells as disclosed below.

An IL27/IL4 compound (e.g., polypeptide construct, fusion protein) of the disclosure is designed. SEQ ID NO: 11 is joined to SEQ ID NO: 24 using the SEQ ID NO: 38 linker, resulting in SEQ ID NO: 48. A poly-histidine tag is added to the N-terminus, and the construct is produced by transient transfection of HEK293E cells as disclosed below.

Additional IL27/IL4 compounds (e.g., polypeptide constructs, fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof) is joined to any one of SEQ ID NOs: 24-45 (or a variant, derivative, or fragment thereof), either directly or via a linker as disclosed herein (for example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The polypeptide constructs (e.g., fusion proteins) are designed in both orientations, e.g., with IL4 located on the C-terminal side of IL27, or with IL4 located on the N-terminal side of IL27. An affinity tag is added to the N-terminus and/or the C-terminus of each construct, and the constructs are produced by transient transfection of HEK293E cells as disclosed below.

An IL33/IL4 compound (e.g., polypeptide construct, fusion protein) of the disclosure is designed. SEQ ID NO: 11 is joined to SEQ ID NO: 26 using the SEQ ID NO: 38 linker, resulting in SEQ ID NO: 49. A poly-histidine tag is added to the N-terminus, and the construct is produced by transient transfection of HEK293E cells as disclosed below.

Additional IL33/IL4 compounds (e.g., polypeptide constructs, fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof) is joined to any one of SEQ ID NOs: 26-32 (or a variant, derivative, or fragment thereof), either directly or via a linker as disclosed herein (for example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The polypeptide constructs (e.g., fusion proteins) are designed in both orientations, e.g., with IL4 located on the C-terminal side of IL33, or with IL4 located on the N-terminal side of IL33. An affinity tag is added to the N-terminus and/or the C-terminus of each construct, and the constructs are produced by transient transfection of HEK293E cells as disclosed below.

An TGFβ1/IL4 compound (e.g., polypeptide construct, fusion protein) of the disclosure is designed. SEQ ID NO: 11 is joined to SEQ ID NO: 34 using the SEQ ID NO: 38 linker, resulting in SEQ ID NO: 50. A poly-histidine tag is added to the N-terminus, and the construct is produced by transient transfection of HEK293E cells as disclosed below.

Additional TGFβ1/IL4 compounds (e.g., polypeptide constructs, fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof) is joined to any one of SEQ ID NOs: 33-34 (or a variant, derivative, or fragment thereof), either directly or via a linker as disclosed herein (for example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The polypeptide constructs (e.g., fusion proteins) are designed in both orientations, e.g., with IL4 located on the C-terminal side of TGFβ1, or with IL4 located on the N-terminal side of TGFβ1. An affinity tag is added to the N-terminus and/or the C-terminus of each construct, and the constructs are produced by transient transfection of HEK293E cells as disclosed below.

An TGFβ2/IL4 compound (e.g., polypeptide construct, fusion protein) of the disclosure is designed. SEQ ID NO: 11 is joined to SEQ ID NO: 36 using the SEQ ID NO: 38 linker, resulting in SEQ ID NO: 51. A poly-histidine tag is added to the N-terminus, and the construct is produced by transient transfection of HEK293E cells as disclosed below.

Additional TGFβ2/IL4 compounds (e.g., polypeptide constructs, fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof) is joined to any one of SEQ ID NOs: 35-37 (or a variant, derivative, or fragment thereof), either directly or via a linker as disclosed herein (for example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The polypeptide constructs (e.g., fusion proteins) are designed in both orientations, e.g., with IL4 located on the C-terminal side of TGFβ2, or with IL4 located on the N-terminal side of TGFβ2. An affinity tag is added to the N-terminus and/or the C-terminus of each construct, and the constructs are produced by transient transfection of HEK293E cells as disclosed below.

TABLE 10 Examples of compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure SEQ ID NO: SEQUENCE 46 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTSPGQGTQSENSCTH FPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 47 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTSPGPVPPSTALRELIE ELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRM LSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN 48 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTFPRPPGRPQLSLQEL RREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTF QAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLD LRDLQRHLRFQVLAAGFNCPEEEEEEEEEEEEERKGLLPGALGSALQGPA QVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQP 49 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTMKPKMKYSTNKISTA KWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETT KRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGI SPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHP SNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFF VLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET 50 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTALDTNYCFSSTEKNC CVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALY NQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 51 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTALDAAYCFRNVQDN CCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSL YNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS

Additional IL4/IL4 compounds (e.g., polypeptide constructs, fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof) is joined to anyone of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment thereof), either directly or via a linker as disclosed herein (for example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The polypeptide constructs (e.g., fusion proteins) are designed in both orientations, e.g., with IL4 located on the C-terminal side of TGFβ2, or with IL4 located on the N-terminal side of TGFβ2. An affinity tag is added to the N-terminus and/or the C-terminus of each construct, and the constructs are produced by transient transfection of HEK293E cells as disclosed below.

IL4-containing fusion proteins of the disclosure are produced by transient transfection of HEK293E cells. Cells are transfected with a pUPE expression vector containing a transgene coding one of the IL4-containing fusion protein sequences. To enable purification, a hexa-histidine affinity tag is cloned at the N-terminus of each IL4-containing protein. Six days post transfection, conditioned medium containing recombinant protein is harvested by low-speed centrifugation (10 minutes, 1000×g) followed by high-speed centrifugation (10 minutes, 4000×g).

Proteins are purified via His-tag by Immobilized Metal Affinity Chromatography (IMAC). In short, the recombinant protein is bound to 0.5 ml Nickel Sepharose® excel at 20° C. Nickel Sepharose® excel containing bound protein is harvested by centrifugation and transferred into a gravity flow column. Non-specifically bound proteins are removed by washing the column with IMAC buffer (500 mM Sodium Chloride, 25 mM Tris, pH=8.2) containing 0 and 10 mM imidazol. The proteins are eluted with IMAC buffer containing 500 mM imidazol. Fractions of 2.5 ml are collected. Recombinant protein-containing fractions are pooled. Conditioned medium and the unbound IMAC fraction are analyzed by LabChip® capillary electrophoresis. The IMAC pool is concentrated to 2-4 ml using an Amicon 10 kDa spin filter. Aggregates are removed by centrifugation (10 minutes 18000×g, 4° C.).

The proteins are purified further by gel filtration using a Superdex200 16/600 column that has been equilibrated in PBS. Protein containing fractions are analyzed by LabChip® capillary electrophoresis and recombinant protein containing fractions are pooled. Protein pools are sterilized by filtration using a 0.22 μm syringe filter and the product stored in 1 ml vials at 80° C.

Protein assays: Protein concentration in batches is determined spectrophotometrically by measuring the absorbance at 280 nm (DropSense16, Trinean) and using a BCA (Thermo Scientific) protein assay.

SDS-Page: Purified proteins are analysed on 12% polyacrylamide SDS-Page gels (Bio-Rad) and bands are visualized by InstantBlue® protein stain (Expedeon; Cambridge).

Example 9: Effects of Compounds (e.g., Polypeptide Constructs, Fusion Proteins) of the Disclosure on Neuronal Sensitization

Experiments are conducted to determine whether IL4-containing compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure affect neuronal sensitization. Cultured neurons are treated with TNFα (50 ng/ml) with and without a compound (e.g., polypeptide construct, fusion protein) of the disclosure, (e.g., at approximately 3 nM). TNFα increases the magnitude of capsaicin-induced calcium influxes compared to untreated cells. Co-treatment with a compound (e.g., polypeptide construct, fusion protein) of the disclosure reduces the sensitization of capsaicin-evoked calcium influx by TNFα. Similar experiments are conducted to determine how sensitivity to PGE2 is affected. Additional experiments are conducted to determine whether the compound of the disclosure affects capsaicin-induced calcium responses in non-sensitized neurons. Constructs are identified that reduce neuronal sensitization by inflammatory mediators. For each polypeptide construct, controls are included to determine the effect of the individual cytokines, and the combination of the cytokines, that the compound (e.g., polypeptide construct, fusion protein) comprises. Constructs are identified that exhibit superiority over individual cytokines, and the combination of cytokines.

Example 10: Compounds (e.g., Polypeptide Constructs, Fusion Proteins) of the Disclosure Crosslink Cytokine Receptors

Experiments are conducted to determine whether compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure crosslink cytokine receptors. Sensory neurons are incubated with polypeptide constructs of the disclosure or the combination of the respective cytokines for 15 minutes in vitro followed by a proximity ligation assay (PLA) to assess receptor clustering. Constructs are identified that induce crosslinking of cytokine receptors.

Example 11: Compounds (e.g., Polypeptide Constructs, Fusion Proteins) of the Disclosure Elicit Distinct Kinase Activity Profiles Compared to the Combination of Unlinked Cytokines

Experiments are conducted to determine whether compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure elicit distinct kinase activity profiles compared to the combination of unlinked cytokines.

Persistent inflammatory pain is induced in mice as using carrageenan as disclosed herein. PAMgene kinase activity profiling is performed to assess global protein tyrosine kinases (PTK) and serine/threonine kinases (STK) activity in homogenates of lumbar DRGs of mice after administration of protein constructs of the disclosure, or their component cytokines in combination. Kinomic profiles are assessed at 30, 60 and 240 minutes after intrathecal administration with either the polypeptide construct, the combination of cytokines, or PBS. To predict the putative upstream kinases, differential phosphorylation profiles are loaded into PhosphoNET, and pathway analysis is conducted. Compounds (e.g., polypeptide constructs, fusion proteins) are identified that elicit different kinomic profiles to the combination of their component cytokines. Differentially phosphorylated substrates, differentially active upstream kinases, and differentially active pathways are identified.

Example 12: Compounds (e.g., Polypeptide Constructs, Fusion Proteins) of the Disclosure Induces Distinct Transcriptomes Compared to the Combination of Unlinked Cytokines

Experiments are conducted to determine whether compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure elicit distinct transcriptomic profiles compared to the combination of unlinked cytokines.

Persistent inflammatory pain is induced in mice as using carrageenan as disclosed herein. RNA sequencing of DRGs is done six hours after administering a compound of the disclosure, or their component cytokines in combination. Principal component analysis (PCA) and hierarchical clustering of the top differentially regulated genes, and pathway analysis are conducted. Compounds (e.g., polypeptide constructs, fusion proteins) are identified that elicit different transcriptomic profiles to the combination of their component cytokines. Differentially expressed genes and pathways are identified.

Example 13: In Vivo Assessment of Compounds (e.g., Polypeptide Constructs, Fusion Proteins) of the Disclosure

Assays are conducted to assess whether the IL4-containing compounds (e.g., polypeptide constructs, fusion proteins) can treat, for example, pain, chemotherapy-induced polyneuropathy (CIPN) pain, nerve fiber loss, and allodynia. The assays are conducted for any compound (e.g., polypeptide construct, fusion protein) disclosed herein, for example, polypeptide constructs comprising an IL4 and a regulatory cytokine, e.g., an IL10/IL4, IL13/IL4, IL27/IL4, IL33/IL4, TGFβ1/IL4, TGFβ2/IL4, or IL4/IL4 of the disclosure.

To induce transient chemotherapy-induced polyneuropathy (CIPN), paclitaxel (2 mg/kg, Cayman Chemical Company) is injected intraperitoneally into C57BL/6 mice on days 0 and 2. To induce persistent paclitaxel-induced CIPN, paclitaxel (8 mg/kg, Cayman Chemical Company) is injected intraperitoneally on day 0, 2, 4 and 6. To induce persistent oxaliplatin-induced polyneuropathy, mice receive two treatment cycles, each consisting of 5 daily intraperitoneal injections of 3 mg/kg oxaliplatin (Tocris) with a 5 days free interval. To induce inflammatory hyperalgesia, mice receive an intraplantar injection of 20 μl λ-carrageenan (2% (w/v), Sigma-Aldrich) dissolved in saline solution (NaCl 0.9%) in both hind paws.

Noxious mechanical sensitivity in the hind paws is measured using von Frey hairs (Stoelting, Wood Dale, USA). Results are expressed as the 50% paw-withdrawal threshold using the up-and-down method. Thermal hyperalgesia is assessed by determining the heat withdrawal latency times using the Hargreaves test (IITC Life Science). In some experiments the length of intraepidermal nerve fibers in the paw skin at day 15 is determined by immunofluorescent staining of skin biopsies with the neuronal marker PGP9.5. All experiments are performed in a blinded manner.

IL4-containing compounds (e.g., polypeptide constructs, fusion proteins) are administered to mice, e.g., via intrathecal injection under light isoflurane/O2 anaesthesia, or by another route as disclosed herein. The ability of IL4-containing compounds (e.g., polypeptide constructs, fusion proteins) of the disclosure to inhibit paclitaxel or oxaliplatin-induced neuropathy, carrageenan-induced inflammatory hyperalgesia, and intra-epidermal nerve fibre loss, is determined and compared to the combination of unlinked cytokines for each construct.

Example 14: Neuroprotective Effects of Compounds (e.g., Polypeptide Constructs, Fusion Proteins) of the Disclosure

Assays are conducted to assess whether the IL4-containing compounds (e.g., polypeptide constructs, fusion proteins) possess neuroprotective properties, e.g., inhibit paclitaxel-induced reduction of neurite length. The assays are conducted for any compound (e.g., polypeptide construct, fusion protein) disclosed herein, for example, polypeptide constructs comprising an IL4 and a regulatory cytokine, e.g., an IL13/IL4, IL10/IL4, IL13/IL4, IL27/IL4, IL33/IL4, TGFβ1/IL4, TGFβ2/IL4, or IL4/IL4 of the disclosure.

Culture of DRG neurons: DRGs are cultured as described previously (Nat. Commun. 4, 1682 (2013)). Briefly, DRGs are dissected and placed on ice-cold dissection medium (HBSS w/o Ca2+ and Mg2+, 5 mM HEPES, and 10 mM glucose). After dissection, axons are cut and dissection medium is replaced by filtered enzyme mix (HBSS without Ca2+ and Mg2+, 5 mM HEPES, 10 mM glucose, 5 mg/ml collagenase type XI (Sigma), and 10 mg/ml Dispase (Gibco)). The DRGs are incubated in enzyme mix for 30 minutes at 37° C. and 5% C02. Subsequently, enzyme mix is inactivated with heat-inactivated fetal bovine serum (FBS, Sigma). Cells are cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% FBS (Gibco), 2 mmol/L glutamine (Gibco), 10,000 IU/ml penicillin-streptomycin (Gibco) on poly-L-lysine (0.01 mg/ml, Sigma) and laminin (0.02 mg/ml, Sigma)-coated glass coverslips in a 5% CO2 incubator at 37° C. Cells are used the following 1-2 days.

Treatments and neurite length measurement: After 24 h in culture, DRG neurons are treated with Paclitaxel (1 μM) alone, or in the presence of different concentrations of IL4 compounds (e.g., polypeptide constructs, fusion proteins) (e.g., 0.12 nM, 0.6 nM, or 3 nM), or equimolar doses of the individual cytokines that are present in the constructs for 24h. Neurites are visualised using p3-tubulin staining, whilst the number of sensory neurons is determined using NeuN staining. Pictures are taken and for each picture, the neurite length per sensory neuron is determined. The neurite length is averaged to a single value for each animal and condition. The percentage inhibition of paclitaxel-induced neurite length loss per mouse culture of 1 mouse is calculated according the following formula ((μ_(control)−μ_(paclitaxel))−(μ_(control)−X_(cytokine)))/(μ_(control)−μ_(paclitaxel))*100 (where p is the average neurite length per neuron averaged over all samples and X is the neurite length of each individual sample).

β3-tubulin and NeuN staining: cells are fixed in 4% PFA for 10 minutes, permeabilized with PBS with 0.05% Tween-20, followed by incubation in blocking buffer (1% BSA and 5% Normal donkey serum in PBS with 0.05% Tween-20 and 0.01% triton) for 1 hour. Cells are incubated with rabbit anti-β3 tubulin (Anti-beta III Tubulin antibody-Neuronal Marker, ab18207, 1:1500, Abcam, Cambridge, UK) and NeuN (Anti-NeuN Antibody clone A60, MAB377, 1:500) Sigma Aldrich (Merck), Darmstadt, Germany) overnight at 4° C., followed by washes and incubation with AF488-conjugated donkey anti-rabbit and 568-conjugated donkey anti-mouse secondary antibodies (Thermofisher, 1:500) followed by DAPI (1:5000, Sigma) staining before sections are mounted on slides with FluorSave reagent (Millipore).

Images are taken using an Olympus IX83 microscope (Olympus). Pictures are analysed using CellSens software (Olympus) and ImageJ (NIH), using the NeuralNetrics macro kindly provided by prof. dr. Winnok de Vos (University of Antwerp) (Pani G, De Vos W H, Samari N, de Saint-Georges L, Baatout S, Van Oostveldt P, et al. MorphoNeuroNet: An automated method for dense neurite network analysis. Cytom Part A. 2014 February; 85(2):188-99). Other plugins used are Olympus Viewer plugin (Olympus). Cell sense software is used to automatically count number of neurons based on NeuN staining. Neurite length is determined using the NeuralNetric macro in ImageJ (Fuij).

Example 15. An IL4-Containing Fusion Protein of the Disclosure Elicits a Distinct Kinase Activity Profile Compared to a Combination of Unlinked Cytokines

Animals: All animal experiments were performed in accordance with international guidelines and with prior approval from the University Medical Center Utrecht experimental animal committee. Experiments were conducted with 8-14 weeks old male and female wild type (WT) C57BL/6 mice.

Paclitaxel-induced CIPN: At day 0, 2, 4 and 6 animals were injected intraperitoneally with 8 mg/kg of Paclitaxel (diluted in Cremophor:EtOH 1:1; volume of injection 40 μl/10 g of bodyweight. At day 8, animals received i.t. injections of: IL4/IL13 fusion protein (0.3 μg), IL4+IL13 (0.15 μg each) or Vehicle.

Drugs and administration: The IL4/IL13 was produced by transient transfection of HEK293F cells with the pcDNA3.1-neo expression vector (Invitrogen; Carlsbad, Calif.) with dual CMV promotor. The vector contained two transgenes: cDNA coding for IL4/IL13 fusion protein and cDNA coding beta-galactoside-2, 3-sialyl-transferase to optimize glycan capping with sialic acid. The IL4/IL13 contained a 6-His tag at the N terminus and was purified through HIS-Select Nickel Affinity gel (Sigma). IL4/IL13 concentrations were determined with an IL4 ELISA kit (IL-4 Pelipair ELISA kit; Sanquin) and Bicinchoninic Acid Protein Assay (BCA Pierce Protein Assay Kit, ThermoFisher Scientific). Intrathecal (i.t.) injections of different compounds (5 μl/mouse) were performed as described before (J Neurosci 30, 2138-2149, 2010) under light isoflurane/O₂ anesthesia. The IL4/IL13 (0.3 μg/mouse) or equimolar doses (0.15 μg each/mouse) of recombinant human HEK-produced IL4 (Sigma) and IL13 (2bsciences) were injected intrathecally at day 8 after the first paclitaxel injection.

Kinase Activity Profiling: Animals were killed an hour after intrathecal injection of the IL4/IL13 fusion protein, IL4+IL13 or vehicle, followed by immediate DRG isolation. Lumbar DRGs were homogenized using M-PER mammalian Extraction buffer (Pierce) supplemented with phosphatase and protease inhibitor cocktails (Pierce). Protein concentration was determined using the Bradford assay (Bio-Rad). Kinase activity profiling was performed using the Tyrosine Kinase PamChip® (PTK) Array for Pamstation®12 (PamGene International B.V.). For the PTK array, 7.5 μg of protein lysate per array was used. Image quantification and statistical analysis were performed using BioNavigator® Software (PamGene International B.V.). Upstream kinase analysis was performed using BioNavigator® Software with peptide-kinase mapping using Kinexus phosphonet enrichment files (www_phosphonet.ca/).

Results

To elucidate downstream signaling in sensory neurons in an unbiased manner, PamGene kinase activity profiling was performed to assess global protein tyrosine kinases (PTK) activity in homogenates of lumbar DRGs isolated from mice with persistent paclitaxel-induced CIPN after IL4/IL13 fusion protein, IL4+IL13 (combination of unlinked cytokines), and vehicle administration. Kinomic profiles were assessed at 60 minutes after intrathecal administration of the IL4/IL13 fusion protein, the combination of cytokines, or vehicle (PBS). Naive mice (i.e. not treated with paclitaxel or IL4/IL13 fusion protein) were also included.

FIG. 11 illustrates peptides that were differentially phosphorylated based on one-way ANOVA analysis between IL4/IL13, IL4+IL13, and vehicle-treated mice compared to naive mice (untreated; no paclitaxel, no intrathecal injection). Black indicates no significant changes, while color indicates decreased phosphorylation. Analyses of the peptides that were differentially phosphorylated by PTK in the DRG homogenates of IL4/IL13-treated versus IL4 plus IL13-treated mice and vehicle-treated mice, indicated that in total 19 peptides were uniquely phosphorylated upon treatment with the fusion protein.

Analyses of the peptides that were differentially phosphorylated by PTK in the DRG homogenates of IL4/IL13-treated male and female mice versus IL4 plus IL13-treated mice, indicated that in both sexes the activity of different kinases is differentially affected by the IL4/IL13 compared to the combination of cytokines (FIG. 12 and FIG. 13).

In DRGs from female mice treated with the IL4/IL13 fusion protein, the activity of several kinases was reduced when compared female mice treated with the combination of IL4 and IL13 (FIG. 12). The graph shows the predicted upstream kinases inferred from the differentially phosphorylated peptide substrates on the PamChips® identified by unpaired t-test comparison between samples from IL4/IL13 fusion protein-treated females and IL4+IL13-treated females (n=3 animals per group). The top 5 predicted putative kinases affected (according to summation of sensitivity and specificity score) were: ITK, RET, TYK2, FER and ERBB4.

In male mice, unique kinase activity was predominantly increased (FIG. 13). The graph shows predicted upstream kinases that can be inferred from the differentially phosphorylated peptide substrates on the PamChips® identified by unpaired t-test comparison between samples from IL4/IL13 fusion protein-treated males and IL4+IL13-treated males (n=3 animals per group). The top 5 predicted putative kinases affected were: LTK, RYK, ALK, AXL and BLK.

These data show that IL4/IL13 uniquely regulates sets of kinases compared to the combination of unlinked IL4 plus IL13.

CONCLUSIONS

In the present work the inventors consider that the coupling of IL4R binding region and a cytokine receptor (e.g., IL10R, IL13R, IL27R, IL33R, TGFβ1R, TGFβ2R) binding region in a single molecule/protein creates a novel molecule that is able to target regulatory cytokine receptors in sensory neurons to trigger unique signalling pathways (e.g., pain resolution pathways) that are not activated by the combination of both wild-type cytokines. Sensory neurons and other cells in the dorsal root ganglion and spinal cord express functional cytokine receptors (e.g., IL4R, IL10R IL13R, IL10R, IL33R, TGFβ1R, and TGFβ2R), and cross-linking these receptors by a compound (e.g., polypeptide construct, fusion protein) or a bispecific antibody or any other compound that can bind at least two of these receptors in sensory neurons, can be used to stop, for example, chronic pain, either nociceptive, neuropathic or combined nociceptive-neuropathic pain. Furthermore, this unique signalling induced by cross-linking of receptors of regulatory cytokines on neurons and glial cells, prevents neuronal damage. 

1-256. (canceled)
 257. A method of normalizing a response of a sensitized human neuron to a stimulus, the method comprising contacting the sensitized human neuron with a compound that comprises an interleukin 4 polypeptide attached to a cytokine, wherein the compound is present at a concentration of at least 1 pM during the contacting, wherein the response of the sensitized human neuron to the stimulus is normalized relative to before the contacting.
 258. The method of claim 257, wherein the compound normalizes the response of the sensitized human neuron to the stimulus to a greater degree than a ten-fold higher concentration of the interleukin 4 polypeptide and the cytokine that are not attached to each other.
 259. The method of claim 257, wherein the compound is present at a concentration of at most 100 μM during the contacting.
 260. The method of claim 257, wherein the human neuron has been sensitized by a pro-inflammatory mediator.
 261. The method of claim 260, wherein the pro-inflammatory mediator comprises a prostaglandin.
 262. The method of claim 260, wherein the pro-inflammatory mediator comprises an inflammatory cytokine.
 263. The method of claim 257, wherein the stimulus induces neuronal action potential firing.
 264. The method of claim 257, wherein the stimulus comprises a pro-inflammatory mediator, a drug, a toxicant, or a chemical stimulus.
 265. The method of claim 257, wherein the response to the stimulus is not significantly altered in a non-sensitized human neuron contacted with the compound.
 266. The method of claim 257, wherein the response comprises a magnitude of a calcium flux response.
 267. The method of claim 257, wherein the response comprises depolarization.
 268. The method of claim 257, wherein the response comprises action potential frequency.
 269. The method of claim 257, wherein the response comprises a signal transduction response.
 270. The method of claim 257, wherein the response is normalized as demonstrated by a lower magnitude of a calcium flux response to a predetermined amount of capsaicin.
 271. The method of claim 257, wherein the contacting decreases ectopic neuronal activity relative to before the contacting.
 272. A method of modulating signaling in a human nervous system cell, the method comprising contacting the human nervous system cell with a compound that comprises an interleukin 4 polypeptide attached to a cytokine under conditions that induce heterologous clustering of a receptor for the interleukin 4 polypeptide and a receptor for the cytokine.
 273. The method of claim 272, wherein the heterologous clustering of the receptor for the interleukin 4 polypeptide and the receptor for the cytokine is as determined by a protein proximity assay.
 274. The method of claim 272, wherein the heterologous clustering of the receptor for the interleukin 4 polypeptide and the receptor for the cytokine is as determined by a proximity ligation assay
 275. The method of claim 272, wherein the receptor for the interleukin 4 polypeptide and the receptor for the cytokine are clustered within 51 nm of each other.
 276. The method of claim 272, wherein the heterologous clustering of the receptor for the interleukin 4 polypeptide and the receptor for the cytokine is not induced by equivalent concentrations of the interleukin 4 polypeptide and the cytokine that are not attached to each other.
 277. The method of claim 272, wherein the heterologous receptor clustering reduces sensitization of the human nervous system cell.
 278. The method of claim 272, wherein the heterologous receptor clustering protects the human nervous system cell against neurotoxicity as determined by a neurite outgrowth assay.
 279. The method of claim 272, wherein the heterologous receptor clustering protects the human nervous system cell against neurotoxicity as determined by an intraepidermal nerve fiber density assay.
 280. The method of claim 272, wherein the heterologous receptor clustering decreases ectopic neuronal activity.
 281. The method of claim 272, wherein the cytokine is a regulatory cytokine.
 282. The method of claim 272, wherein the heterologous receptor clustering induces an altered kinomic profile compared to a human nervous system cell that is contacted with equivalent amounts of the interleukin 4 polypeptide and the cytokine that are not attached to each other as determined by kinase array profiling.
 283. The method of claim 282, wherein altered kinomic profile comprises altered JAK-STAT signaling.
 284. The method of claim 283, wherein a level of activity of JAK1, c-Kit, or c-Met is modulated. 